Current Topics in Membranes and Transport, Volume 04

Current Topics in Membranes and Transport Vdllllls 4 Advisory Board Robert W . Berliner Britton Chance I . S. Edelma...

Author: Felix Bronner | A. Kleinzeller

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Current Topics in Membranes and Transport Vdllllls 4

Advisory Board

Robert W . Berliner Britton Chance I . S. Edelman Aharon Katchalsky (deceased) Adam Kepes Richard D. Keynes Philip Siekevitz Torsten Teorell Daniel C. Tosteson Hans H . Ussing

Contributors

Richard P. Durbin Mahendra Kumar Jain Howard E. Morgan Carolyn W . Slayman Carol F . Whitjeld

Current Topics in Membranes and Transport

VOLUME 4

Edited by Felix Bronner Department of Oral Biology University of Connecticut Health Center Farmington, Connecticut and

Arnost Kleinzeller Graduate Division of Medicine University of Pennsylvania Philadelphia, Pennsylvania

1973

Academic Press

New York and London

A Subsidiary of Harcourt Brace Jovanovich, Publishers

COPYRIGHT 0 1973, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMllTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

ACADEMIC PRESS, INC.

111 Fifth Avenue, New York, New York 10003

United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD. 24/28 Oval Road, London NWI

LIBRARYOF

CONGRESS CATALOQ

CARD

NUMBER:

PRINTED IN THE UNITED STATES OF AMERICA

70-117091

List of Contributors, vii Preface, ix Contents of Previous Volumes, xi Aharon Katzir-Katchalsky, 1913-1972, xiii Bibliography of the Principal Publications of Aharon Katzir-Katchalsky on Membrane Phenomena, xix The Genetic Control of Membrane Transplant CAROLYN W. SLAYMAN

I. Introduction, 1 11. Isolation of Transport Mutants, 3 111. Criteria for Identifying Genes That Affect Transport Directly, 136 IV. Linkage Relationships of Transport Mutants, 140 V. Dominance and Recessiveness, 146 VI. Regulation of Transport Systems, 150 VII. Usefulness of Mutants in Understanding Transport Mechanisms, 151 VIII. Conclusion, 155 References, 155 Enzymic Hydrolysis of Various Components in Biomembranes and Related Systems MAHENDRA KUMAR JAIN

I. Introduction, 176 11. Action of Hydrolytic Enzymes on Model Systems, 184 111. Effect of Enzymic Hydrolysis on System Properties of Biomembranes, 191 IV. Effect of Enzymic Hydrolysis on Transport Systems, 217 V. Catabolism of Membrane Components by Endogenous Enzymes or Intracellular Catabolism, 233 VI. Conclusions and Epilog, 236 References, 238 Regulation of Sugar Transport in Eukaryotic Cells HOWARD E. MORGAN AND CAROL F. WHITFIELD

I. Kinetic Characterization of Passive Transport, 256 11. Nonhormonal Regulation of Sugar Transport, 261 111. Hormonal Control of Sugar Transport, 274 IV. Mechanisms of the Regulation of Transport, 287 V. Summary, 296 References, 297 V

CONTENTS

Vi

Secretory Events in Gastric Mucosa

RICHARD P. DURBIN I. Introduction, 305 11. Comparative Aspects, 305 111. Structural Aspects, 307 IV. Coupling of Secretion to Metabolism, 313 V. Conclusion, 319 References, 319 Author Index, 323 Subject Index, 340

List of Contributors Cardiovascular Research Institute and Department of Physiology, University of California, San Francisco, California Mahendra Kumar Join, Department of Chemistry, Indiana University, Bloomington, Indiana* Howard E. Morgan, Department of Physiology, The Milton S. Hershey Medical Center, The Pennsylvania State University, Hershey, Pennsylvania Carolyn w. Slayman, Departments of Human Genetics, Microbiology, and Physiology, Yale University School of Medicine, New Haven, Connecticut Carol F. Whitfleld, Department of Physiology, The Milton S. Hershey Medical Center, The Pennsylvania State University, Hershey, Pennsylvania Richard P. Durbin,

* Present address: Department of Chemistry, University of Delaware, Newark, Delaware. vii

This Page Intentionally Left Blank

The fourth volume of Current Topics in Membranes and Transport extends the analysis of significant transport processes and structures. Carolyn Slayman presents a comprehensive summary of the genetics of transport, Jain reviews some aspects of the bilayer nature of the biological membrane, Morgan and Whitfield deal with sugar transport and its control in eukaryotes, and Durbin reviews some problems of gastric ion secretion. The editors hope that all interested in biological transport will find these reviews rewarding and provocative. Last year we dedicated the volume to the memory of Aharon KatairKatchalsky. Caplan’s thoughful appreciation in this volume reminds us of the breadth of Aharon’s work and of the human qualities that went into it. We hope this series will continue to reflect the scientific ideal of Aharon Katdr-Katchalsky, namely a passionate belief in the dispassionate search for scientific truth. FELIX BRONNER ARNOST KLEINZELLER

lx


Contents of Previous Volumes Volume 1

Some Considerations about the Structure of Cellular Membranes MAYNARD M. DEWEYAND LLOYDBARR The Transport of Sugars across Isolated Bacterial Membranes H. R. KABACK Galactoside Permease of Escherichia coli ADAMKEPES Sulfhydryl Groups in Membrane Structure and Function ASERROTHSTEIN Molecular Architecture of the Mitochondrion DAVIDH. MACLENNAN Author Index-Subject Index Volume 2

The Molecular Basis of Simple Diffusion within Biological Membranes W. R. LIEBAND W. D. STEIN The Transport of Water in Erythrocytes ROBERTE. FORSTER Ion-Translocation in Energy-Conserving Membrane Systems B. CHANCE AND M. MONTAL Structure and Biosynthesis of the Membrane Adenosine Triphosphatase of Mitochondria ALEXANDER TZAGOLOFF Mitochondria1 Compartments : A Comparison of Two Models HENRYTEDESCHI Author Index-Subject Index

xi

xii

CONTENTS OF PREVIOUS VOLUMES

Volume 3

The Na+, K+-ATPase Membrane Transport System: Importance in Cellular Function E. LINDENMAYER, AND ARNOLD SCHWARTZ, GEORGE JULIUS C. ALLEN Biochemical and Clinical Aspects of Sacroplasmic Reticulum Function ANTHONY MARTONOSI The Role of Periaxonal and Perineuronal Spaces in Modifying Ionic Flow Across Neural Membranes W. J. ADELMAN,JR.AND Y. PALTI Properties of the Isolated Nerve Endings GEORGINA RODR~GUEZ DE LORESARNAIZAND EDUARDO DE ROBERTIS Transport and Discharge of Exportable Proteins in Pancreatic Exocrine Cells : In Vitro Studies J. D. JAMIESON The Movement of Water Across Vasopressin-Sensitive Epithelia RICHARD M. HAYS Active Transport of Potassium and Other Alkali Metals by the Isolated Midgut of the Silkworm AND KARLZERAHN WILLIAMR. HARVEY Author Index-Subject Index

Aharon Katzir-Katchalrky


Aharon Katzir-Katchalsky, 1913-1972 To write an objective biographical appraisal of Aharon Katchalsky’s contribution to the membrane field, with the recollection of his brutal and senseless death a t the hands of political assassins still raw, is scarcely possible. It is too early, and for one who knew him as a friend, and fell under his spell as a teacher, too painful. Every aspect of his work is colored by memories and associations: the elation he could evoke a t a seminar or discussion around the coffee table; the sense of excitement-sometimes bordering on euphoria-he never failed to communicate to the audience during his lectures; the intense pleasure he took (and gave) in debating a theoretical point, chalk in hand. He loved elegance and style wherever he found them, but especially in a page of mathematics; a blackboard covered with equations in his own graceful handwriting often possessed something of the richness of a medieval manuscript. Of what manner of a man he washis warmth, humanity, and largeness of spirit-others have written eloquently. For those of us who had found inspiration in his teaching, including his oldest colleagues, he always remained in the profoundest sense of the word a mentor. In a way it is an invidious task to separate Katchalsky’s studies on membranes from the totality of his work. Viewed as a whole, his work is as impressive in its unity as it is astonishing in its variety. The twin themes of mechanochemical coupling and chemodiffusional coupling dominate large parts of it, both being intimately related to the most characteristic properties of biological systems. His contributions to the understanding of such processes do not, however, stand apart from his contributions to polymer chemistry and nonequilibrium thermodynamics. His ideas on membranes encompassed many dimensions of the problem and had wide ramifications. The earliest papers, written in the late ~ O ’ S ,were directed towards the proper understanding of existing experimental techniques and the proper interpretation of permeability measurements commonly made on biological membranes. The implications were speedily realized to have importance in the technological application of membranes-especially in desalination. For Katchalsky, however, synthetic membranes remained models for biological systems, and by the mid-60’s the problem of the coupling of chemical reaction and transport had become paramount in his thoughts. In his last years he was deeply concerned with the mechanism of memory recording, which involved considerations relating to hysteretic effects in both biopolymers and biomembranes. As always the thermodynamic implications of the phenomena intrigued him, and frequently led him far into the realm of philosophical speculation. Katchalsky’s early work on membranes and transport phenomena in general was a natural outgrowth of his studies, covering more than a decade, of the chemical physics and biophysics of macromolecules. At the outset of his career he was profoundly impressed by the notion that large polymeric molecules might play an essential role in all processes occurring in living systems-as a consequence of, among other things, their conformational degrees of freedom. This was a t a time when little was known of protein structure and DNA was still to be recognized as the carrier of genetic information. However, the work of Staudinger and later Kern in the 30’s had suggested that charged synthetic polymers would be useful as models of biological macromolecules. I n the brief period between the end of World War I1 and the beginning of the Israeli War of In-

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AHARON KATZIR-KATCHALSKY, 1 9 1 3-1 972

dependence, Katchalsky developed his ideas during a seminal spell in the laboratory of the redoubtable Werner Kuhn in Basel, and returned to Palestine (as it then was) to found a school of polyelectrolyte chemistry a t the Hebrew University on Mount SCOPUS. With the conclusion of hostilities this school shifted to Rehovot, where he was called upon to establish a polymer department a t the newly-created Weizmann Institute of Science. Starting with papers in the first volumes of the Journal of Polymer Science in 1946 and 1947,Katchalsky and his colleagues produced a veritable avalanche of theoretical and experimental publications, making an explosive impact on the rapidly growing field of polyelectrolyte solutions and gels. The impact of these studies was also felt in the equally rapidly developing technology of ion-exchange resins and membranes, stimulating the concerns which led ultimately to Katchalsky’s later work on the permeation of salt through charged membranes. In the papers written during this “polyelectrolyte” period the seeds of his deep and lasting interest in mechanochemistry are to be found, and the profound influence of Gibbsian thermodynamics on his thinking is already strikingly in evidence. In the early 50’s Staverman demonstrated the utility of nonequilibrium thermodynamics in describing membrane processes, and introduced the concept of a reflection coefficient in relation to the osmometry of polymer solutions. Kirkwood showed that local force equations (written in terms of resistance coefficients), in contrast to local flow equations, may be integrated across a membrane to give global phenomenological relations. But very few workers appreciated the power of the method until 1958, when Kedem and Katchalsky, and also Spiegler, published almost simultaneously and quite independently their classical papers on the application of nonequilibrium thermodynamics to membrane transport. Kedem and Katchdsky were concerned primarily a t that time with the permeability of biological membranes to nonelectrolytes; Spiegler waa concerned with transport processes in ion-exchange membranes. Katchalsky had always understood the importance of thermodynamics as an organizing principle, often claiming that it “plays the role of scientific logics.” It was therefore characteristic of his thinking to invoke nonequilibrium thermodynamics when his attention turned to membranes and transport. Undoubtedly the contribution for which Katchalsky is chiefly known (and may well be chiefly remembered) among workers in the membrane field is precisely his collaborative study of permeability with Kedem, which led to the derivation of what they called “practical” phenomenological equations. These Kedem-Katchalsky (or K-K) equations already possess a time-honored air. As Richardson has pointed out, it is little over a decade since the most complete versions of the equations were publbhed, yet they have already taken their place alongside the Nernst-Planck and the Goldman equations as standard working models for physiologists and biophysicists. I n fact the KedemKatchalsky equations are familiar to a generation of young biophysicists who may never read the original papers. In one simple form, applicable to systems involving a single solute, the equations describe volume flow (Jy)and solute flow (J.)through a homogeneous membrane in the absence of electric current:

J.

=

c,(l

- u)JV + COAT

(2)

Here A p and AT are the hydrostatic and osmotic pressure differences respectively, and cs is an average of the solute concentrations in the solutions on either side of the membrane. The “practical” phenomenological coefficients appearing in Eqs. (1) and (2) are the filtration coefficient L,, the solute permeability U , and the reflection coefficient Q

xvii

AHARON KATZIR-KATCHALSKY, 1913-1 972

originally introduced by Staverman. Prior to the derivation of these equations there existed no self-consistent treatment of permeability, and certainly none general enough to cover the whole range of physiological phenomena. Conventional descriptions of membrane transport made use of two independent flow equations, one for volume and one for solute, with only two coefficients. Thus volume flow was represented by an expression of the type

Jv = Lp (Ap - AT) and solute flow by the Fickian form

both manifestly incomplete in the light of present-day concepts, since the possibility of coupling between flows is entirely ignored. Although experimentalists had become progressively aware of the inadequacy of expressions such as these, and although nonequilibrium thermodynamic treatments of transport were in the air and indeed osmotic pressure measurement had been characterized in terms of the reflection coefficient, it remained for the Kedem-Katchalsky equations to link the phenomena, for the first time, into a single gestalt capable of yielding an internally coherent description in physiological terms. This literally revolutionized membrane studies. Equation (2) indicates that the solute permeability w is to be determined under condi. an alternative version tions of zero volume flow, by measuring the ratio ( J s / A ~ ) ~ v 4 In of the equations a “second permeability” w‘ appears, to be determined under conditions such that the hydrostatic and osmotic pressure differences just balance, i.e., by measI. version can be written conveniently in a form uring the ratio ( J a / A ~ ) ~ 9 - ~This such that the all-important property of Onsager symmetry (identity of the cross-coefficients) is displayed:

J,

- A T ) + ca(l - U ) L ~ ( A T / C J - a ) L P ( A p- A T ) + C ~ W ’ ( A T / C J

= Lp(Ap

J a = c,(l

(3) (4)

Equations (1) and (3) are identical, and the last term in Eq. (4) is obviously just o’Aa. Clumsy though they are, these expressions bring out the nature of the thermodynamic forces conjugate to the flows J, and J., which are seen to be ( A p - AT) and ( A r / c . ) respectively. Such conjugate flux-force pairs are generated, according to the methodology of nonequilibrium thermodynamics, by first deriving the so-called “dissipation function,” i.e., the temperature times the rate of entropy production due to irreversible processes in the membrane. This takes the form

T(diS/dt) = J v ( A p

- AT)

J&a0

(5)

where the quantity Apec denotes the concentration-dependent part of the chemical potential difference of the solute across the membrane. Equation ( 5 ) is exact for dilute solutions providing that local processes within the membrane obey linear relations. Kedem and Katchalsky sought to elicit, from the global phenomenological relations corresponding to this dissipation function, a set of transport coefficients which to some extent would have the virtue of familiarity to researchers in the field (and thus be readily accessible experimentally and compatible with existing data), to some extent would be insensitive to concentration changes, but above all would be thermodynamically selfconsistent. To reconcile these requirements they were compelled to introduce the

xviii

AHARON KATZIR-KATCHALSKY, 191 3-1 972

appropriate average concentration csr such that Apse = A a j c ,

(6)

Consequently, cs is a logarithmic average. This transformation of the dissipation function has the virtue of “saving the phenomenon,” and led directly to the formulation of the practical flow equations. It is perhaps a reflection of the importance attached to the Kedem-Katchalsky equations that they did not fail to attract their share of criticism. It has been variously claimed that they are misleading on a t least two grounds: that their range of validity (the linear regime) must be vanishingly small, and that global Onsager symmetry cannot actually hold when bulk flow occurs. This is not the place to analyze such criticisms in depth, but a comment seems to be called for. It is basic to the nonequilibrium thermodynamic approach that the forces be small enough to ensure linearity of the flux equations. The transformation from ApBcto AT is itself nonlinear since it invokes the logarithmic average concentration-but it is the key to the entire representation. Certainly this limits the applicability of Eqs. (1) and (2) to very small values of J , or A T , but it does not by any means render them invalid (especially for the description of physiological membrane processes). On the contrary, it has been shown repeatedly that the KedemKatchalsky equations represent a first-order expansion of the integrals of the local frictional equations; indeed the expansion indicates that when the concentration ratio exceeds (say) 2:1, so that c8 departs by more than a few percent from the arithmetic average, use of the latter can preserve the linear formalism if J , is not too large. I t also emerges from such calculations that the reflection coefficients appearing in Eqs. (1) and ( 2 ) [or Eqs. (3) and (4)]are identical, and hence global Onsager symmetry does in fact obtain, a result which has been verified experimentally in several systems. But clearly it is in the nature of linear phenomenological equations that they represent behavior within certain limits-they are not expected to be exact under all circumstances. However small the range of the Kedem-Katchalsky relations may be, their heuristic and practical importance remains unquestionably immense. Perhaps the turning point in the realization by biologists of the significance of the new approach was the memorable symposium on membrane transport and metabolism held in Prague in 1961, when by all accounts Katchalsky electrified the audience by his presentation. At any rate, from then on the biological literature reflects a growing interest in the formalism. Hard on the heels of this meeting appeared an interpretation of the practical phenomenological coefficients in terms of friction and distribution coefficients. Here Kedem and Katchalsky turned their attention to charged membranes and demonstrated that for electrolyte permeation through such membranes, the reflection coefficient u may assume negative values, indicating “anomalous” osmosis. The mechanism of anomalous osmosis had been a controversial topic: its elucidation in phenomenological terms was thus a vivid demonstration of the scope of the method. The analysis was performed for a membrane conforming to the well-known Teorell-MeyerSievers fixedcharge model, yielding results in good agreement with the experimental data of Loeb and of Grim and Sollner. This study led naturally to the consideration of electric current flow through charged membranes. In an elegant treatment it was shown that if the potential across the system is applied by means of electrodes reversible to one of the ions present, the dissipation function requires just one more term-current times the potential difference between the electrodes. A fully developed treatment of the permeability of highly charged membranes t o electrolytes was presented in a series of three successive papers by Kedem and Katchalsky in 1963. These papers analyzed the properties of composite membranes in terms of series

AHARON KATZIR-KATCHALSKY, 1 9 1 3-1 9 7 2

xix

and parallel arrays, emphasizing the fundamental importance of polarity and circulation (respectively) in understanding the behavior of such structures. I n particular, this analysis showed that mosaic membranes may exhibit pronounced negative anomalous osmosis, i.e., high negative values of u, an effect which had been predicted qualitatively much earlier by Sollner and demonstrated by Neihof and Sollner. The substance of Katchalsky’s work on membrane and other transport processes up to this point was incorporated in the book “Nonequilibrium Thermodynamics in Biophysics” by Katchalsky and Curran, published in 1965. I n the late 60’s Katchalsky became increasingly absorbed in the problem of coupling between diffusion and chemical reaction, especially as manifested in biological membranes. An examination of the thermodynamics and kinetics of active transport in erythrocytes (with Blumenthal and Ginzburg) was followed by an analysis of facilitated diffusion (with Blumenthal) which modeled, inter a h , the allosteric transitions of a carrier protein. At this time periodicity in membranes assumed an important place in his thinking-especially periodicity related to the presence of chemical reaction. Together with Spangler he showed that if an autocatalytic system undergoes a phase transition with metastable states, oscillatory behavior can be achieved by appropriately coupling the reaction t o a membrane transport process regulating the flow of product or reactant. These considerations invoked the concept of thermodynamically metastable ateady states and suggested the possibility that hysteresis loops might exist in transitions between steady states. Such hysteresis cycles in biomembranes were linked t o similar phenomena in biopolymers and t o the phenomenon of memory. I n 1969 Katchalsky wrote the following credo*:

I believe that the ultimate goal of biological study is to “translate” the phenomena of life into meaningful physical concepts. It is rather clear that present-day physics is still unable t o deal adequately with the complex and diversified expressions of life, and many years of research and contemplation await the scientists before they will be able to fit animate and inanimate matter into a common, unified conceptual framework. The driving force of quantitative biological study is, however, our mystical conviction that “Nature” is one and that future generations will comprehend life within an integrated “Natural Philosophy of the Physical World.” The profound belief in the unity of nature expressed here led Katchalsky t o pondex deeply the chemical basis of morphogenesis and the origin of life. His ideas were influenced primarily by the early work of Turing on the ability of homogeneous chemical processes to develop structure spontaneously (as a consequence of a random disturbance) and the more recent work of Prigogine on the thermodynamic theory of structure and stability. Structures which survive only by the dissipation of an energy input were termed by Prigogine “dissipative structures.” They can appear in systems maintained far from thermodynamic equilibrium, and are characterized by an unstable transition point (a “symmetry-breaking” transition or instability). Such transitions in chemical systems require nonlinear reaction schemes such as occur in the autocatalytic process referred to above. Katchalsky considered that dissipative structures may not only play a major role in the maintenance of certain cellular patterns, but that they may also have participated in the development of the earliest structures from which life arose. Prior to his end Katchalsky was preoccupied with several complementary interests: the nature of prebiotic peptide synthesis, the consequences of chemodiffusional coupling,

* I n “Biology and the Physical Sciences” (S. Devons, ed.). Columbia Univ. Press, New York.

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AHARON KATZIR-KATCHALSKY, 1 9 1 3-1972

and the basis of the molecular memory record. Through all these there runs as a unifying thread the notion of generation, storage, and retrieval of information. This notion is strongly reflected in his later writings concerning membrane phenomena. He viewed chemodiffusional coupling as a structuring agent closely related to biological morphogenesis, and this led him to speculate that active membranes might be dissipative structures whose dynamics are governed by the very chemodiffusional processes participating in their action. Indeed he conjectured, in agreement with Prigogine, that all living organization may be based on dissipative structures, fixed to dxerent degrees by covalent bonds. His deep concern with information flow, energy flow, and the establishment of pattern and shape led him to break fresh ground in the application of thennodynamics to complex systems. A love of thermodynamic rigor expressed itself in everything he did, and his final major work-which was cut short even as it began to flourish-was an attempt to develop a “network thermodynamics” especially suited to the organizational complexity of biological systems. By generalizing network theory to include irreversible thermodynamic systems, thermodynamics was to be brought within the framework of modern dynamic systems analysis. The bond graph approach to systems analysis, still in its infancy, impressed Katchalsky with its versatility as a representation of arbitrarily complex networks. In his last paper on this subject, completed just before he died, he showed, together with Oster and Perelson, that the bond graph technique can be used to describe diffusion-reaction systems including facilitated and active transport, the rectification properties of complex membranes, and relaxation oscillations in coupled membrane systems. Many of Katchalsky’s ideas on these and other matters live on in the minds of his colleagues, and will ultimately see the light of day. But although the direction in which the broader thrust of his thinking lay may be known, we are denied the pleasure of ever witneesing its realization. His work will be carried forward, but we shall never know which path Katchalsky himself would have trodden. The particular flavor and style that was his will be missing, and so will the insight and the flair for finding connections among a bewildering variety of seemingly unrelated concepts. Katchalsky was born in Lodz, Poland, came to Israel in 1925, and received his M.Sc. and Ph.D. degrees from the Hebrew University in 1937 and 1940. He had many honors, and engaged in a host of activities. He was the first President of the Israel National Academy of Sciences and Humanities, President and then Honorary Vice-president of the International Union for Pure and Applied Biophysics, a Council Member of the International Council of Scientific Unions, a Foreign Member of the United States National Academy of Sciences, a Council Member of the European Molecular Biology Organization, and Visiting Miller Professor of the University of California a t Berkeley. He was a true Renaissance man, a citizen of the world, renowned for his charismatic charm, his encyclopedic knowledge of topics ranging from history to philosophy and metaphysics, his love of the cut and thrust of scientific dialogue. In his work he constantly emphasized historical perspective, and in his passing he symbolized the historic confrontation of his people-creativity versus hatred and destruction. The cruel snuffing out of his life deprived us all of a very precious source of enlightenment, but he left a legacy of riches to build on for a long time.

S. R. CAPLAN

Bibliography of the Principal Publications of Aharon Katzir-Katchalsky on Membrane Phenomena Kedem, O., and Katchalsky, A. (1958). Thermodynamic Analysis of the Permeability of Biological Membranes to Non-electrolytes. Biochim. Biophys. Acta 27, 229. Katchalsky, A. (1961). Membrane Permeability and the Thermodynamics of Irreversible Processes. “Membrane Transport and Metabolism” (A. Kleinzeller and A. Kotyk, eds.), p. 69. Academic Press, New York. Kedem, O., and Katchalsky, A. (1961). A Physical Interpretation of the Phenomenological Coefficients of Membrane Permeability. J . Gen. Physiol. 45, 143. Katchalsky, A,, and Kedem, 0. (1962). Thermodynamics of Flow Processes in Biological Systems. Biophys. J . 2, 53. Kedem, O., and Katchalsky, A. (1963). Permeability of Composite Membranes. Part I. Electric Current, Volume Flow and Flow of Solute Through Membranes. Trans. Faraday SOC.59, 1918. Kedem, O., and Katchalsky, A. (1963). Permeability of Composite Membranes. Part 11. Parallel Elements. Trans. Faraday SOC.59, 1931. Kedem, O., and Katchalsky, A. (1963). Permeability of Composite Membranes. Part 111. Series Array of Elements. Trans. Faraday SOC.59, 1941. Ginaburg, B. Z., and Katchalsky, A. (1963). The Frictional Coefficients of the Flows of Nonelectrolytes through Artificial Membranes. J . Gen. Physiol. 47, 403. Katchalsky, A., and Curran, P. F. (1966). “Nonequilibrium Thermodynamics in Biophysics,” Harvard Univ. Press, Cambridge, Massachusetts. Blwnenthal, R., Ginzburg, B. Z., and Katchalsky, A. (1967). Thermodynamic and Model Treatment of Active Ion Transport in Erythrocytes. “Hemorheology” (Proc. First Intern. Conf.), p. 91, Pergamon, New York. Katchalsky, A. ( 1967). Membrane Thermodynamics. “The Neurosciences, A Study Program” (G. C. Quarton, T. Melnechuk, and F. 0. Schmitt, eds.), p. 326. The Rockefeller Univ. Press, New York. Katchalsky, A., and Spangler, R. (1968). Dynamics of Membrane Processes. Quart. Rev. Biophys. 1, 127. Katchalsky, A. ( 1968). Thermodynamic Treatment of Membrane Transport. Pure Appl. Chem. 16,229. Katchalsky, A. (1968). Thermodynamic Consideration of Biological Membranes. “Membrane Models and the Formation of Biological Membranes” (L. Bolis and B. A. Pethica, eds.), p. 318. North-Holland, Amsterdam. Blumenthal, R., and Katchalsky, A. (1969). The Effect of the Carrier AssociationDissociation Rate on Membrane Permeation. Biochim. Biophys. Acta 173, 367. Katchalsky, A. (1969). Membrane Thermodynamics. “Membranes $. Permeabilite SBlective,” p. 19. Editions du Centre National de la Recherche Scientifique, Paris. Katchalsky, A. ( 1969). Non-equilibrium Thermodynamics of Bio-Membrane Processes. “Theoretical Physics and Biology” (M. Marois, ed.), p. 188. North-Holland, Amsterdam. Katchalsky, A., and Oster, G. (1969). Chemico-Diffusional Coupling in Biomembranes. xxi

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AHARON KATZIR-KATCHALSKY, 1 9 1 3-1 972

“The Molecular Basis of Membrane Function” (D. C. Tosteson, ed.), p. 1. PrenticeHall, Englewood Cliffs. Katchalsky, A. (1970). Thermodynamic Consideration of Active Transport. “Permeability and Function of Biological Membranes” (L. Bolis, A. Katchalsky, R. 1). Keynes, W. R. Loewenstein, and B. A. Pethica, eds.), p. 20. North-Holland, Amsterdam. Katchalsky, A. (1971). Thermodynamics of Flow and Biological Organization. ZYGON: J . Relig. Sci. 6, 99. Katchalsky, A. (1971). Biological Flow Structures and Their Relation to ChemicoDiffusional Coupling. Neurosci. Res. Prog. Bull. 9, 397. Oster, G., Perelson, A., and Katchalsky, A. (1971). Network Thermodynamics. Nature (London) 234, 393. Katchalsky, A., and Neumann, E. (1972). Hysteresis and Molecular Memory Record. Int. J . Neurosci. 3, 175. Oster, G. F., Perelson, A. S., and Katchalsky, A. (1973). Thermodynamics of Biological Networks. Quart. Rev. Biophys. 6, 1.

The Genetic Control of Membrane Transport CAROLYN W. SLAYMAN Departments of Human Genetics. Microbiology. and Physiology. Yale Univwsily School of Medicine. New Haven. Connecticut

. .

Introduction . . . . . . . . . . . . . . . . . . Isolation of Transport Mutants . . . . . . . . . . . . A Resistance t o Analogs . . . . . . . . . . . . . . B Selection of Nongrowing Cells . . . . . . . . . . . C Direct Screening for Uptake . . . . . . . . . . . . D . Recognition of Transport Defects in Higher Organisms . . . . E . The Strategy of Recovering Transport Mutants . . . . . . F. The Problem of Multiple Transport Systems for a Single Substrate . I11. Criteria for Identifying Genes that Affect Transport Directly . . . . IV Linkage Relationships of Transport Mutants . . . . . . . . A . Escherichia coliand Salmonella typhimurium . . . . . . . B . Neurospora crassa . . . . . . . . . . . . . . . V Dominance and Recessiveness . . . . . . . . . . . . . VI . Regulation of Transport Systems . . . . . . . . . . . . VII . Usefulness of Mutants in Understanding Transport Mechanisms . . . A . In Determining the Number of Separate Transport Systems for a Particular Substrate . . . . . . . . . . . . . . B In Identifying the Components of a Transport System . . . . C . In Determining the Functionof a Component . . . . . . . VIII . Conclusion . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . .

I I1

. . .

.

.

.

1 3 3 129 132 133 133 134 136 140 140 144 146 150 151

151 152 152 155 155

1. INTRODUCTION

The genetic approach to the study of membrane transport can be an extremely useful one. By isolating and mapping mutants defective in the transport of a particular substrate. it is possible to determine the number of systems involved and even the number of subunits in each system; by analyzing the kinetics and the biochemistry of transport in the mutants. 1

2

CAROLYN W. SLAYMAN

one can often obt,ain information about molecular mechanisms. Progress in both of these areas is discussed in this article, together with some of the problems that remain. Membrane genetics is still a relatively young field. The first realization that transport systems, like simple cytoplasmic enzymes, are under genetic control came in the 1950s with work on cystinuria in man and lactose transport in Escherichia coli. Cystinuria had long been known to be an inherited disease, and in fact was one of the “inborn errors of metabolism” (together with pentosuria, alkaptonuria, and albinism) discussed by Archibald Garrod in his famous Croonian lectures in 1908. By 1951 cystinuria had been shown to involve the excretion of abnormal amounts of arginine, lysine, and ornithinc, as well as cystine. The structural relationship among this group of amino acids led Dent and Rose (1951) to postulate that the disease was caused by a primary defect in transport across the renal tubules. Since that time cystinuria has been found to affect the intestinal mucosa as well, and the transport defect has been well characterized i n vitro in intestinal tissue obtained by biopsy (Thier et al., 1964, 1965; McCarthy et al., 1964). In addition, other inherited diseases have been shown to lead to transport defects in man (for example, Hartnup disease, iminoglycinuria, methionine malabsorption, tryptophan malabsorption, X-linked hypophosphatemia), and the idea that all mammalian transport systems are under genetic control-even though mutations may often be lethal or otherwise undetectable-is now widely accepted. The genetic analysis of transport in microorganisms also dates to the 1950s. Although the earlier results of Davis and of Doudoroff had drawn attention to the cryptic nature of certain enzyme systems in bacteria (in which intact cells were unable to metabolize a given substrate even though cell-free extracts contained the necessary enzymes), it was the work of Cohen and Rickenberg (1955) and Rickenberg et al. (1956) that first demonstrated the existence of a specific bacterial transport system. These investigators showed that the product of the lacy gene is required for the transport of p-galactosides in E . coli, and that its synthesis is regulated jointly with the synthesis of the enzyme 8-galactosidase. The kinetics of the transport system have since been examined in detail, both in wild-type E. coli and in lacy mutants, and more recently the l a c y gene product ([%I protein”) has been isolated by Fox and Kennedy (1965). In microorganisms, and particularly in E . coli, this initial work has been followed by the isolation of many different kinds of transport mutants, and the notion that transport systems are genetically determined has been amply documented. This article discusses tfhetechniques that have been developed to isolate

GENETIC CONTROL OF MEMBRANE TRANSPORT

3

and characterize transport mutants, the criteria for identifying genes that affect transport directly, the linkage relationships of transport mutants in several organisms (E. coli, Salmonella typhimurium, Neurospora crassa), the regulation of synthesis of transport systems, and the usefulness of mutants in understanding transport mechanisms. I n .order to simplify the discussion, basic information about the properties of existing transport mutants has been collected in Table I, leaving the text free to consider the general topics just listed. [For further information on microbial transport systems, the reader is referred to recent reviews by Heppel (1971), Kaback (1970a, b, 1972), Lin (1970, 1971), Oxender (1972a, b), Roseman (1972), and Simoni (1972); and for a more complete description of genetic defects affecting transport in man, to reviews by Rosenberg (1969), Rosenberg and Scriver (1969), Scriver (1969), Thier and Alpers (1969), Scriver and Hechtman (1970), and to several chapters in Stanbury et al. (1972).] II. ISOLATION OF TRANSPORT MUTANTS

As interest in the genetic analysis of transport has increased, a variety of methods havc been developed for the isolation of transport mutants, particularly in microorganisms. Some are selection methods, making use of conditions under which the transport mutant can grow but the wild-type cell cannot (or the mutant survives and the wild type is killed); others are merely screening methods, permitting the rapid identification of transport mutants among large numbcrs of wild-type cells. The particular strategy to be used in a given instance depends, as discussed below, on the function of the transport system in question and on whether or not there are alternate routes for the substrate to enter the cell. A. Resistance to Analogs

One of the most powerful methods involves selecting mutants whose growth is resistant to an appropriate analog. This approach has been particularly successful in the isolation of amino acid, purine, and pyrimidine transport mutants (Table 11), but has also been used for carbohydrate, cation, and anion transport mutants. Cclls can, of course. become resistant to analogs through several kinds of mutations-not only those reducing the uptake of the analog, but very frequently those affecting later steps in metabolism (see review by Umbarger, 1971). In studying the effects of D-cycloserine (an analog of alanine) in Streptococcus strain Challis, for example, Reitz et al. (1967) found two classes of resistant mutants. The first possessed a defective

TABLE I Mutations Affecting Membrane Transport Amino Acids and Peptides Organism

Transport System

Specificity

Escherichia coli

Aspartate

A constitutive, high-affinity transport system which is fairly specific f o r L-aspartate (K, = 3.7 x 10-6M I . Ki's for D-aspartate, L-glutamate, L-glutamine, and a series o f aspartate analogs (N-formyl-L-aspartate, N-methyl-DL-aspartate, a-methyl-DL-aspartate, p-methyl-DL-aspartate, DL-eryfhro-p-hydroxyespartate, and DL-fhreo-p-hydroxyaspartate) are substantially higher, ranging f r o m 1.8 t o 8.4 x lo4 M (Kay, 1971 ).

asf

C4 Dicarboxylic acids (aspartate, fumarate, malate, succinate).

A n inducible, low-affinity system which takes u p aspartate, fumarate, malate, maleate, and succinate w i t h Km's o f 10 t o 30 x 10-6M. (Kay and Kornberg, 1971;Loefal., 1972).

dct

Glutamate

A transport system, partially repressed in wild-type E. coli (Marcus and Halpern, 1969).which is fairly specific for L-glutamate (K, = 7.7 x 10-6M; Halpern and Even-Shoshan, 1967).Competitively inhibited by D-glutamate, L-glutamine, and several glutamate derivatives (L-glutamate-y-methyl and -y-ethyl esters, p-hydroxy-DL-glutamate, a-methyl -DL-glutamate) w i t h Ki's o f 2.10 x 10-5 t o 3.35 x 10-3M (Halpern and Even-Shoshan, 1967).Shows complex kinetics w i t h nonlinear double reciprocal plots under some

4

Gene

glfC

Amino Acids and Peptides Linkage

Near xyl (Kay and Kornberg, 1969; Lo etal., 1972). May be allelic with the FMmutant of E. coli. isolated by its inability to grow on succinate and later shown to be deficient in the uptake of dicarboxylic acids and to map near x y l (Herbert and Guest, 1970).

Method of Isolating Mutants

Transport Defect in Mutants

Resistance to DL-rhreo-phydroxyaspartaw (in E. coli K-12; Kay, 1971). An unsuccessful attempt was made to isolate ast mutants from a dct strain (which lacks the general dicarboxylic acid transport system; see below) by selecting for the inability to use aspartate as sole nitrogen source; several mutants with this phenotype were found but proved t o transport aspartate as well as the parent strain (Kay, 1971).

Lack the high-affinity aspartate transfor aspartate port system. The V,,, uptake in one ast mutant, HA3, was decreased from 39 to 25 nmolesl minute per milligram dry weight, and and a single Km (30 x 1 0 6 MI was observed, compared with two Km's (39 and 3.7 x 10-6M) in the wildtype. The defect is also seen in isolated membrane vesicles from ast mutants (Kay, 1971).

Resistance to 3-fluoromalate (Kay and Kornberg, 1969; Lo era/., 1972) or L(-)-tartrate (Kay and Kornberg, 1971). dct mutants are also unable to grow on malate, succinate, or fumarate as sole carbon source (Kay and Kornberg, 1969, 1971). Revertants, selected for the ability t o grow on any one of the C4 dicarboxylic acids, have simultaneously recovered the ability to grow on a l l the other acids (Kay and Kornberg, 1969,1971).

Lack tho low-affinity dicarboxylic acid uptake system. I n one dct for aspartate mutant, F M l , the V,, uptake was decreased to 1.5 inmolesl minute per milligram dry weight, and there was a single K, of 3.5 x 10-6M (Kay, 1971).

dct mutants must be distinguished from strains with a primary block in the tricarboxylic acid cycle; such strains often showa secondary impairment of the ability to take up one or more C4 acids (Kay and Kornberg, 1971). Between ma and pyrE (Marcus and

Halpern, 1969).

Isolated, in wild-type E. coli W, H,or K-12 (site), by the ability to grow o n glutamate as carbon source (Halpern and Umbarger, 1961; Halpern and Lupo, 1965; Marcus and Halpern, 1967).g/tcC mutants also show increased sensitivity to 2methyl-DLglutamate (Halpernand Umbarger, 1961).

gltc is believed to be the operator for gltS (see below).gltC mutants show quantitative but not qualitative alterations in glutamate transport; in a series of such strains the V,, was increased by a factor of 2 to 7, while the K,,, was unchanged (Halpern and Lupo, 1965; Marcus and Halpern, 1969). Furthermore,gltC andgltS are very closely linked, and gltC mutants

(Con tinued) 5

TABLE I Mutations Affecting Membrane Transport (Continued) Amino Acids and Peptides Oraanism

TransDort Svstem

Escherichia coli IC0n't.l

SDecificitv

Gene

conditions; interpreted in terms of an allosteric model in which succinate, aspartate, a-ketoglutarate, yaminobutyrate, or glutamate can activate transport by binding a t a second site (Halpern and EvenShoshan,

1967). A glutamate-binding protein, released from g l C mutants during spheroplast formation, is thought to be involved in glutamate transport. It has a KD of 6.7 x 1 0 6 M (close to the K, of the transport system), is inhibited competitively by L-glutamate-y-methyl ester and noncompetitively by alanine, and can restore the capacity of spheroplasts for glutamate uptake (Barash and Halpern, 1971).

g/fS

gltR

Glutamine

A highly specific transport system for glutamine (K, = 0.8 x l o 7 M ) ,competitively inhibited by y-glutamylhydrazide and y-glutamylhydroxamate but not by any naturally occurring amino acids. Transport is thought to involve a glutamine-binding protein, which is released by osmotic shock and has a K D (for glutamine) of 3 x M. Both transport and the binding protein are repressed by growth in a rich medium (Weineret a/., 1971; Weiner and Heppel,

-

1971).

-

6



Transport Defect in Mutants are derepressed (with high levels of glutamate transport activity) even in the presence of a normal g/tR gene (the postulated repressor gene; see below). The kinetics of transport in gitCC strains are discussed further in Halpern (19671. Halpern and EvenShoshan (1967). and Frank and Hopkins (1969).

Closely linked to g/tC (see above; Marcus and Halpern, 1969).

Isolated, from agltCc parent strain (see above), by loss of the ability to grow on glutamate (Marcus and Halpern, 19691. Might also be isolated from gltCc by resistance to 2methyl-D L-glutamate (Halpern and Umbarger, 1961; Halpern and Lupo, 1965).

g/tS i s thought to be the structural gene for the glutamate transport system.g/rS mutants with qualitatively altered transport systems have been isolated; in strain CS 7/50, for e x ample, the Vmax was decreased by a factor of 3,and the K,,, was increased by a factor of 20 (from 5 x 10.6 M to 1 x 1 0 4 M ) (Marcusand Halpern, 1969). No mutants with altered allosteric properties (see Specificity) have yet been reported.

Near met.4; not linked to gltc and g/tS (Marcus and Halpern, 1969).

Temperature-sensitive g/tR mutants were isolated, from wild-type E. coli, by their ability to grow on glutamate at 42' but not a t 30" (Marcus and Halpern, 1969).

g/tR i s thought to be the repressor gene for the glutamate transport operon. In one temperature-sensitive g/tR mutant (CS ZTC), the V,,, of transport a t 42' was increased by a factor of 4.5, with no change in Km. Furthermore, brief periods of heating a t 44' in the absence of growth increased the differential rate of synthesis of glutamate transport activity during subsequent growth a t 30°, suggesting that the repressor is thermolabile in this mutant (Marcus and Halpern, 1969).

Ability to grow on glutamine as sole carbon source (Weiner e t al.. 1971; Weiner and Heppel, 1971).

Increased transport of glutamine. Mutant strain GLNP 1 showed a 3-fold higher initial uptake rate (with a normal Km) and had three times more binding protein than the parent strain (Weiner and Heppel, 1971).

Resistance to y-glutamylhydrazide (Weiner and Heppel, 1971).

Decreased transport of glutamine. In mutant strain GH 20, both the initial uptake rate and the amount of binding protein were decreased by about 90% (and the small amount of uptake

7

(Continued)

TABLE I Mutations Affecting Membrane Transport (Continued) Amino Acids and Peptides Organism

Transport System

Specificity

Gene

Escherichia

coli (Con%)

Basic amino acids (arginine, lysine, ornithine)

argf Wild-type E. coli K-12 has three kinetically distinct transport systems for basic amino acids (Rosen, 1971a):

(1) a specific, high-affinity system for arginine (K,,, = 2.6 x lo-* M ) ; (2) a specific system for lysine (K, = 1 x 10-5 M ) , inhibited by thiosine; and

(3)a general system (LAO) for lysine (K, = 0.5 x 10-6 M ) , ornithine (K,,, = 1.4 x 10-6 M ) ,arginine, and canavanine. Osmotic shock causes the release of several argininebinding proteins, one of which may play a role i n the arginine-specific transport system (Wilson and Holden, 1969a, b; Rosen, 1971a). and i n addition a lysinearginine-ornithine-binding protein (LAO), which appears to be associated with the general transport system. The L A O protein has a molecular weight of 30,000 and KD's of 3.0 x 10-6 M for lysine, 1.5 x 1 0 - 6 M for arginine, and 5.0 x 10'6M for ornithine (Rosen, 1971a). The lysine-specific system i s not affected by osmotic shock under the usual conditions. I n cells in which the general system has been repressed, however, lysine transport becomes sensitive to osmotic shock and a lysine-binding (LS) protein i s released. This protein is labile at 4" and is rapidly inactivated at ionic strengths above 0.02 (Rosen, 1971b).

8


Method o f Isolating Mutants

Transport Defect i n Mutants that remained may have been mediated by another system, since it was inhibited by glutamate). The genetic relationship between strains GLNP 1 and GH 20 has not yet been established, but the quantitative correspondence b t w e e n uptake rates and amounts o f binding protein in the two strains has been used to support the idea that binding i s involved in transport (Weiner e r a / . , 1971; Weiner and Heppel, 1971 ).

Near serA (Taylor, 1970).

Resistance t o canavanine (€. coli W, Schwartz e t a l . , 1959; E. coli K - I 2, Maas, 1965; Rosen, 1971a).

Canavanine-resistant ( a r g f ) mutants are generally defective in the transport of all three basic amino acids (Scwartz e r a / . , 1959; Maas, 1965; Rosen, 1971a; Maas. cited i n Rosen, 1971a), b u t the exact function o f the argP locus i s not clear. Rosen (1971a) reported, in one such mutant (Can R22). that two transport systems were affected; arginine transport was completely missing, and ornithine and lysine transport via the L A O system were partially reduced. Both the arginine-specific binding protein and the LAO-binding protein have now been found t o be present i n Can R22, and the mutant shows normal facilitated diffusion of arginine (assayed by coupling arginine uptake to arginine decarboxylase, and measuring COP production) (Rosen. personal communication). Rosen has therefore postulated that the lesion i s in energy coupling, presumably in a factor common t o both the arginine-specific and L A O transport systems. However, Maas (personal cornmunication) found that another argP mutant, JC 182-5, produces an altered LAObinding protein with a lowered binding capacity for arginine, ornithine, and lysine; the arginine-specific binding proteins appear normal i n JC 182-5. Further work will be

9

(Continued)

TABLE I Mutations Affecting Membrane Transport Konrinuedl Amino Acids and Peptidm Organism

Transport System

Specificity

Gene

Eschsrichie coli ICon'rJ

Aromatic amino acids (tryptophan, tyrosine, phenylalanine)

Wild-type E. coli K-12 has at least f i w transport systems for the aromatic amino acids (Brown, 19701:

aroP

(1) a general aromatic system for tryptophan (Km = 4.0 x lO-7M). tyrosine (K, = 5.7 x l o 7 M1,and phenylalanine (K,,, = 4.7 x 10-7 M I, with much lower affinities for several other amino acids (histidine, leucine, methionine, alanine, cysteine, and aspertate). The general system is inhibited by p-fluorophenylalanine, @-2-thienylalanine,and 5-methyltryptophan; (2) a specific system for tyrosine (K,,, = 2.2 x 1 0 - 6 ~ ) . inhibited by p-fluorophenylalanine (and sewral other analogs) and b y high concentrations of phenylelanine;

(3)a specific system for phenylalanine (K,,,

= 2.0 x

10-6 M I , similarly inhibited by p-fluorophenylalanine (and several other analogs) and by high concentrations of tyrosine; (41 a specific system for tryptophan (K, = 3.0 x 10-6 M I , inhibited by 4methyltryptophan (and other analogs); and

(5) an inducible system for tryptophan (Boezi and DaMoss, 1961 ; Burrous and DeMoss, 1963).

10

trpf



Transport Defect in Mutants required to identify the primary effect of the argP gene on the LAO system (whether on binding, energy coupling, or both), and t o learn why arginine-specific transport i s affected in some argP mutants (Can R22) but apparently not in others (JC 182-5).

Between leu and pan (Brown, 1970).

Resistance to thiosine (Rosen, personal communication).

Lack the specific transport system for lysine but contain the lysinebinding protein (Rosen, personal communication).

Resistance to p-2-thienylalanine (E. coli K-12; Brown, 1970). The aroP mutants were also resistant to p-fluorophenylalanine and 5-rnethyltryptophan, and additional aromatic transport mutants might be isolated using these analogs.

Lack the general aromatic transport system (Brown, 1970).aroP mutants retain the specific tryptophan, tyrosine, and phenylalanine systems, with K;s similar to those of the wild-type strain but with somewhat lower V,,,'s. This latter finding, if significant, may mean that the general and specific transport systems share the component determined by the gene aroP (Brown. 19711.

Isolated in an aroP mutant of

Not discussed.

E. coli K-12 strain W31 10, which has a high level of the tryptophan-specific transport system, by resistance to 4methyltryptophan (Yanofsky, cited in Oxender, 1972a). Between trpA and ton6 (Thorne and Corwin, 1970).

Resistance to indole acrylic acid (Thorne and Corwin, 1970).

Two lines of evidence have led to the view that a gene for tryptophan transport is located between trpA and ton6 (Thorne and Corwin, 1970, 1971): (1) Deletions in this region cause a 10-fold decrease in tryptophan uptake, and ( 2 ) low uptake in point mutants, selected by resistance to indole acrylic acid, can be restored to normal by introduction of the F'trp episome. However, the uptake of other amino acids was not measured in these experiments, and the results might be explained by the observation that deletions extending into the ton6

11

(Continued)


Transport System

Specificity

Gene

Eschen'chia coli IConW

Histidine

Not characterized.

Glycine. alanine, wine

Wild-type E. coli K-12 has t w o (or three) kinetically distinct transport systems for glycine. atanine, and serine (Wargel etal., 1970; Qxender, 1972a):

-

cyc

(1) a system for L-alanine (Km = 5.7 x 10-5 M ) and probably L-serine. inhibited by L-cycloserine and 0-carbamyl-Dserine; and

(2) and (3?)a system for glycine, D- and L-alanine. and D-serine. with nonlinear double reciprocal plots which have been resolved into t w o sets of Km's and VmaX's (Km's = 2.5 and 9.1 x 10-5 M for glycine, and 2.5 and 8.2 x 1 0 %M for D-alanine). This system is inhibited by D-cycloserine.

Leucine, isoleucine, valine

There appear t o be multiple transport systems for the branched-chain amino acids i n E, coli, but the kinetic picture i s not yet clear. Guardiola and laccarino (1971) reported a single set of Km's for laucine, isoleucine, and valine (7 x 10-6 M, 4.8 x l o 6 M and 12 x l o 6 M, respectively); but Piperno and Oxender (19681observed two K,'s for valine (0.7 and 8 x 1 0 6 MI, Furlong and Weiner (1970) observed t w o Km'S for leucine (0.2 and 2 x 10-6 M I, and more recently 12

brnP brnQ

Amino Acids and Poptades Linkage


Transport Defect i n Mutants region lead t o decreased transport of a variety of amino acids, as i f tun8 influences general membrane permeability (Yanofsky, cited in Oxender, 1972a).

c y c r l (cycA) maps near purA (Russell. 1972); and cyc'2 and cycr3. which are cotransducible with one another, map at least 0.5 minute away (Curtiss et a/., 1965).

Penicillin treatment of a histidine-requiring strain i n low-histidine medium I€.coli W; Lubin eta/., 1960).

Defective i n the uptake of histidine (Lubin e t a / . , 1960).

Resistance t o D-cycloserine; three successive steps in resistance (cycrl, cycr2. and cycr3) have been identified in €. coli K-12 by Curtiss ot a/. (1965). Other investigators have selected by means of resistance to D-cycloserine (E. colt W, Kessel and Lubin, 1965; Wargel et al., 1971) or Dw i n e (E. coli W, Davis and Maas. 1949; Kessel and Lubin, 1965: E. coli K-12, Cosloy and McFall. 1971; Oxender, 1972a), or by penicillin treatment of glycine-requiring strains I€.culi W and B) growing in lowglycine medium (Kessel and Lubin. 1965). The relationship of these mutants t o the cyc loci has not been renorted

Defective in the transport of glycine. D- and L-alanine, and Dderine (Schwartz et a/., 1959; Levine and Simmonds, 1960. 1962; Lubin era!., 1960; Kessel and Lubin. 1965; Kaback and Kostellow. 1968; Kaback and Stadtman, 1968; Wargel et at.. 1971; Oxender, 1972a). Thecycrl strains lack the high-affinity component of transport, and the cycr2 and cycr3 strains lack the lowaffinity component (Wargel er &., 1971).

Isolated, i n a Dserine-resistant mutant that lacked the glycine. D- and L-alanine. D-serine systemls), by penicillin salection on L-alanine (Oxender, 1972a).

brnP maps near leu, and bmQ near phoA (Guardiola and laccarino, 1971).

Resistance t o valine (Guardiola and laccarino, 19711. The growth of wild-type E. coli K-12 i s inhibited by valine, which blocks isoleucine biosynthesis through feedback inhibition o f acetolactate synthetase (Leavitt and Umbarger. 1962). I n addition t o the

13

The double mutant has lost about

95%of the L-alanine transport capacity. and is assumed t o lack the

L-alanine. L-serina system as well as as the glycine. D- and L-alanine, D-serine system (Oxender, 1972al. brnP mutant M I 183a showsd abnormal Km's for isoleucine ( 3 6 x 106M.comparedwith4.8 x 1 0 - 6 M in the parent strain) and for leucine (0.4 and 7 x 1 0 6 M, respectively), although not for valine (12 and 12.5 x 1 0 8 M ) ; Guardiola and laccarino 11971) concluded that it might h a w a qualitatively altered transport sys-

(Continued)


Escherichia coli (Con't.)

Transport System

Specificity

Gene

Guardiola, De Felice, and laccarino (oersonal communication) detected four systems for the branchedchain amino acids, a shared system with K,'s of about 2 x 10.6 M for leucine, isoleucine, and valine, and specific systems for each of the three amino M. acids with Km's of about 50 x At least two binding proteins have been isolated. One has comparable affinities for leucine, isoleucine, and valine, and presumably is associated with the shared LIV transport system. It has a molecular weight of about 36,000, and both i t and LIV transport are repressed when leucine is present in the growth medium (Piperno and Oxender, 1968; Anraku, 1968a, b, c; Nakane eta/., 1968; Penrose eta/., 1968, 19701. The second protein binds only leucine. It is remarkably similar to the first in molecular weight, amino acid composition. and K D for leucine; it, like the first, is repressed by leucine; and the two proteins cross-react immunologically, suggesting a common developmental origin (Furlong and Weiner, 1970; Furlong, personal communication I .

dlu

14

A m i n o Acids and Peptides Linkage

Method of Isolating Mutants valine-resistant mutants defective in transport, others have been described that possess either a valine-resistant acetolactate synthetase or an increased rate o f isoleucine biosynthesis (Glover, 1962; Ramakrishnan and Adelberg, 1964,1965).

Transport Defect in Mutants t e m for the branched-chain amino acids, and thus that brnP might be the structural gene f o r this transport system. By contrast,brnO m u t a n t M I 1 7 4 b f o r isoshowed decreased V,,,'s leucine, valine, and leucine (by a factor of 5 t o 10). and it was suggested that brnO might be a regulatory gene (Guardiola and laccarino, 1971). With the discovery o f multiple transport systems f o r leucine, isoleucine, and valine (see Specificity), however, the kinetic analysis o f b r n P a n d brnO mutants w i l l have to be carried o u t in greater detail before one can conclude which system o r systems are altered b y these mutations and which are unaffected. Recently Guardiola, De Felice, and laccarino (personal communication) measured the amounts and the dissociation constants of b o t h the L I V and the leucine-binding proteins in 6rnP and b r n 0 mutants, and have found the proteins to be normal. They conclude tentatively that brnP and brn0 d o n o t code f o r either binding protein.

Isolated, in a leucine-requiring strain, b y the ability t o use D-leucine as a source o f Lleucine (Rahmanian and Oxender, 1972a.b).

Increased transport o f D- and Lleucine, isoleucine, and valine via the shared system, and increased amounts o f the LIV-binding protein. dlu is concluded t o be a regulatory gene f o r the L I V system (Rahmanian and Oxender, 1972b).

Isolated, in a dlu parent strain (see above), by resistance t o azaleucine (Rahmanian and Oxender, 1972b).

One class o f azaleucine-resistant mutants lacks L I V transport activity b u t retains the LIV-binding protein; another class shows reduced transport activity f o r branched+hain amino acids and also f o r other, unrelated amino acids (Rahmanian and Oxender, 1972b).

15

(Continued)

TABLE I Mutations Affecting Membrane Transport (Continued Amino Acids and Peptides Organism

Transport System

Escherichia

Cystine ,

co/i (Con%) diaminopimelic acid

Specificity Wild-type E. coli W has two transport systems for

Gene

-

cystine: (1) a general system (K,,, = 3 x lo-' M ) , inhibited by diaminopimelic acid (DAP), and (2) a specific system (K, = 2 x 1 0 8 M ) ,not inhibited by DAP. The activity of the general system is reduced by osmotic shock, and a cystine- and DAP-binding protein (molecular weight 28,000; K D for cystine = 2 x l o 7 M ) has been partially purified from the shock fluid (BergereraL, 1971).

Proline

Wild-type E. coli takes up proline with a K,,, of 6.4 x 10-7 M. Uptake is inhibited by azetidine-2-carboxylic acid and 3.4-dehydroproline (with Ki's of 2.4 x 10-5 M, and 2.6 x 1 0 6 M, respectively) and by several other proline analogs (Tristram and Neale, 1968). A prolinebinding activity has recently been partially purified from membrane vesicles (Gordon era/., 1972).

-

Dipeptides

A transport system for dipeptides containing two L-amino acids or glycine plus one L-amino acid (Kessel and Lubin, 1963;Sussman and Gilvarg, 1971).

-

Oligopeptides

A transport system for oligopeptides containing lysine, omithine, glycine, tyrosine, and perhaps other amino acids (Payne, 1968;Sussman & Gilvarg, 1971).

-

16

Amino Acids and Peptidm Linkage



Diaminopimelic acid-requiring mutants of E. coli W normally grow slowli with DAP as sole supplement, and require lysine, i n addition, for normal growth; "D" mutants were isolated which had lost the partial requirement for lysine (Leive and Davis. 19651.

Lack the specific cystine transport system (Berger et a/., 1971).

Penicillin treatment of a proline-requiring strain of €. coli W or B in low-proline medium (Lubin etal., 1960; Kessel and Lubin, 1962).

Defective in proline transport, both in intact cells (Kessel and Lubin, 1962) and in isolated membrane vesicles (Kaback and Stadtman, 1966; Kaback and Deuel, 1969). Normal, however, in the passive diffusion of proline across the membrane (Kessel and Lubin, 1962; Kaback and Stadtman, 1966).

Resistance t o 3.4-dehydroproline or L-azetidine-2-carboxylic acid ( E . coli strain C4; Tristram and Neale, 1968). Al I dehydroprol ine-resistant strains tested showed crossresistance t o azetidine, b u t by contrast the azetidine-resistant strains (14 tested) were sensitive t o dehydroproline.

Some of the dehydroproline-resistant mutants were defective i n proline uptake; others showed normal uptake b u t excreted large quantities of proline (Tristram and Neale, 1968). Most azetidine-resistant mutants were reduced i n proline uptake. One mutant was of particular interest because i t s transport system had normal affinities for proline and dehydroproline but a 10-fold reduced affinity for azetidine. In a few azetidine-resistant mutants, proline uptake was normal, proline was not excreted in large quantities, and the mechanism o f resistance is unknown (Tristram and Neale, 19681. The genetic relationship among the various mutants has not yet been determined.

Penicillin treatment of a glyauxotroph of E. col; W o n glycylglycine medium (Kessel and Lubin, 19631.

Defective uptake of glycylglycine and other dipeptides (Kessel and Lubin, 1963).

Resistance t o triornithine

Indirect evidence (from growth experiments) for a defect i n the transport of oligopeptides (Payne and Gilvarg, 1968; Payne, 1968).

(E. coli W; Payne and Gilvarg, 1968; Payne, 1968).

17

(Continued)

TABLE I Mutations Affecting Membrane Transport (Continued) Amino Acids and Peptides Gene

Organism

Transport System

Specificity

Salmonella typhimurium

Aromatic amino Wild-type S. ryphimurium has at least six transport aroP acids (tryptosystems for the aromatic amino acids (Ames, 1964): phan, tyrosine, ( 1 ) a nonspecific aromatic system which transports phenyla'anine'tryptophan (K, = 5 x 10-7 M),tyrosine (Km = 2 x 10-6 histidine) M),phenylalanine (Km = 5.9 x l o 7 M),and histidine (K, = 1.1 x lo4 M).Inhibited by 2-methylhistidine, 3-pyrazolealanine, 2-thiazolealanine, p-fluorophenylalanine,5-methyltryptophan, p-2-thienylalanine, p-3-furylalanine, azaserine, and the phosphonate derivatives of phenylalanine and tyrosine (Ames, 1964; Ames and Roth, 1968); (2) and (3) at least two specific histidine systems, designated J-P and K-P. with K,'s of M and l o 7 M ,respectively (Arnes and Lever, 1970);

(4)a specific tryptophan system (Ames, 1964); (5)a specific tyrosine system, inhibited b y p-fluorophenylalanine and by high concentrations of phenylalanine (Ames, 1964);and

(6)a specific phenylalanine system ( K , = 2 x which is also inhibited by p-fluorophenylalanine (Ames, 1964).

M),

Recently reviewed by Ames (1974a.b)

hisP

18

~~

Amino Acids and Peptides Linkage Near p r o A (Ames and Roth, 1968).

Method o f Isolating Mutants Resistance t o azaserine (Ames, 1964) or t o tyrosine or phenyl. alanine phosphonate derivatives (Ames and Roth, 1968). Mutants resistant to p-fluorophenylalanine and 5-methyltryptophan were also isolated, but turned out to be phenylalanine excretors rather than transport mutants (Ames,

1964).

Transport Defect i n Mutants Lack the general aromatic transport system. aroP mutants are not very defective in the uptake o f tryptophan, tyrosine, or phenylalanine (since these amino acids are also transported b y the specific systems), but do show greatly reduced uptake of p-fluorophenylalanine. Control experiments, i n which aroP culture supernatant was found t o have no effect on uptake by wild-type cells, ruled out the possibility that the primary defect in aroP was the excretion of some compound that inhibited FPA uptake (Ames, 1964).

An attempt t o isolate specific phenylalanine transport mutants by resistance t o p-fluorophenylalanine (FPA) in the presence of tyrosine (to inhibit the general aromatic transport system) was n o t successful; the mutants that were isolated had normal rates of FPA uptake (Ames,

1964). Near p u r F (Ames and Roth, 1968). Closely linked t o hisJ and dhuA (Ames and Lever, 1970).

Resistance t o 2-hydrazino-3(4-imidazolyl) propionic acid (HIPA) (Shifrin eta/., 1966; Ames and Roth, 1968).

Lack the two specific histidine transport systems, J-P and K-P. Only a small amount of residual histidine transport remains i n hisP mutants, with a K, of about 10-6 M; this residual transport occurs via the general aromatic system and at least two additional low-affinity systems; (Ames and Lever, 1970). Evidence t o be described below indicates that the hisP gene product, known to be a protein because o f the existence of amber mutants (Ames and Roth, 1968). interacts with two binding proteins-J and K-to constitute the specific histidine transport systems. The amount o f the binding proteins is not altered i n hisP mutants (Ames and Lever, 1970).

19

(Contimed)

TABLE I Mutations Affecting Membrane Transport (Continuedl Amino Acids and Peptides Organism

Transport System

Specificity

Gene hisJ

Salmonella typhimorium (Con%I

dhuA

20



Transport Defect i n Mutants

See hisP

Isolated, from a dhuA hisparent strain (see below), b y loss of the ability t o grow on D-histidine while s t i l l retaining sensitivity t o HlPA (Ames and Lever, 1970).

Lack the J-binding protein, which binds D- and L-histidine and HI PA, and interacts with the P protein t o constitute one mode of specific histidine transport (Ames and Lever, 1970). hisJ mutants retain the K-P system, and can transport histidine with a K,, of about 2 x 10-7 M. Evidence that hisJ is i n fact the structural gene for the J-binding protein has come from the study of a strain that contains two mutations in hisJ, both induced by the frameshift mutagen ICR191; the initial mutation caused loss of both the J-P mode of transport and the J protein, and the second mutation caused partial restoration of both. In this strain the J protein is abnormal-it is temperature-sensitive and has altered chromatographic and electrophoretic properties, Concomitantly, histidine transport via the J P system has become temperature-sensitive, reinforcing the idea that the J protein is an obligatory component of the transport system, as well as indicating that hisJ is i t s structural gene (Ames and Lever, 1972). A detailed biochemical characterization of the J protein has recently been reported (Lever, 1972).

See hisP.

Isolated, from a his- auxotroph, by the ability t o use D-histidine as a source of L-histidine (KrajewskaGrynkiewicz eta/., 19711.

Shows a 2- to 3-fold increase i n the transport o f L-histidine, a significant transport o f D-histidine (not seen in the parent strain), and an increased sensitivity t o HlPA (KrajewskaGrynkiewicz eta/., 1971; Ames and Lever, 1970). In addition,dhuA mutants contain a 4- to 5-fold increased level of J-binding protein (which has the same chromatographic properties and binding constant as the protein from wild type) (Ames and Lever, 1970). All these results are consistent with the idea that dhuA i s a regulatory gene for hisJ.

21

(Continued)

TABLE I Mutations Affecting Membrane Transport (Continued) Amino Acids and Peptides Oraanism

TransDort Svstem

Salmonella typhimurium (Con 't.I

Tryptophan

Pseudomona aeruginosa

Specificity

Gene -

Leucine

Not characterized (but note the complexity of the branched-chain amino acid transport systems in E. coli).

Methionine

Wild-type S. typhimurium has two transport systems for methionine, one with an affinity 50 times that of the other. Methionine transport is inhibited at least partially b y the analogs a-methylmethionine, ethionine, norleucine, and methionine sulfoxime, but it has not yet been reported whether these compounds block the high-affinity transport system, the lowaffinity system, or both (Ayling and Bridgeland, cited in Smith, 19711.

Proline

There is kinetic evidence for two proline transport systems in wild-type P. aeruginosa, one saturating below 1 x 10-6 M, and the other above 20 x M. The first, high-affinity system is quite specific for L-proline and is inhibited only by close analogs (thioproline, dehydroproline, L-azetidine-2-carboxylic acid); it is induced by growth in the presence o f proline (Kay and Gronlund, 1969b).

Aromatic amino acids (tryptophan, tyrosine, phenylalaninel

Preliminary evidence suggests that wild-type P. aeruginosa has at least two transport systems for the aromatic amino acids, differing in their relative affinities for tryptophan, tyrosine, and phenylalanine, and also in their sensitivity t o inhibitors (D-phenylalanine,p-fluorophenylalanine, 5- and 6-fluorotryptophan) (Kay and Gronlund, 19711.

22

-

metP

-

Amino Acids and Peptides Linkage Between trp and chr

Method of Isolating Mutants -

This region is thought t o include a gene that affects tryptophan transport (system not specified), since deletion mutants take u p greatly reduced amounts of tryptophan (Thorne and Corwin, 1970; but see discussion of similar mutants in E. cold.

(Thorne and Corwin, 1970).

Near chr, on the side distal t o trp (Thorne and Corwin, 1972).

Maps i n the leu-jwrE region (Ayling and Bridgeland, cited i n Smith, 1971).


Similarly, the trp-chr region is believed t o contain a gene affecting leucine transport, since deletions in this region lead t o a decrease in leucine uptake (Thorne and Corwin, 1972); from a comparison of several deletion mutants, it was concluded that the postulated leucine gene i s not the same as the postulated tryptophan gene. Resistance t o a-methylmethionine sulfoxime (Ayling and Bridgeland, cited in Smith, 1971).

Lack the high-affinity methionine transport system (Ayling and Bridgeland, cited in Smith, 1971).

Slow growth on proline as carbon source (Kay and Gronlund, 1969a).

Defective uptake of proline via the high-affinity system (Kay and Gronlund, 1969a.b).

Slow growth on tryptophan as carbon source (mutant strains TClO and TA3). or resistance t o 5-fluorotryptophan (mutant strain 5FT3). The genetic relationship among these three strains is not certain. 3 0 p fluorophenylalanine-resistant mutants were also isolated, but none was defective in aromatic amino acid transport (Kay and Gronlund, 1971).

Strain TClO was primarily defective in the uptake of tyrosine, strain 5FT3 in the uptake of tryptophan, and strain T A 3 in the uptake of both. I t was suggested that the mutations may have affected, respectively, the postulated tyrosine-preferring system, the postulated tryptophan-preferring system, and a common element of both systems (Kay and Gronlund, 1971). Since kinetic constants have not yet been reported f o r any of the mutants (or for the wild type),

23

(Continuedl


Transport System

Specificity

Gene

Aeudomonas aeruginosa /Con?.)

Pseudomonas fluorescens

Streptococcus faecalis

Arginine

Not characterized.

lsoleucine

Not characterized.

L-Alanine, P-alanine, L-proline

Wild-type P. fluorescens has a common transport system for L-alanine (K, = 1.4 x 1 0 4 M),palanine (Km '6-8 x 1 0 5 M ) , and L p r o l i n e (Km = 2.6 x 1 0 5 M),and in addition two specific transport systems for L-alanine (Km's = 2 and 8 x 10-6 M )end one for Lproline (Km = 5 x 10-6 M ) (Hechtmen and Scriver, 1970a.b).

-

S. faecalis is thought t o take up L-lysine and hydroxyL-lvsine via a specific transport system, and L-lysine, hydroxy-L-lysine, and L-arginine via a general system (Friede etal., 1972). The kinetic constants of the t w o systems have not yet been reported.

-

Wild-type S faecalis has two transport systems for acidic amino acids (Reid etal., 1970):

-

Lysine, arginine

Aspartate, glutamate

( 1 ) a high-affinity (HA) system which transports aspartate (K, = 1 x 10-5 MI, glutamate (K, = 3 x 10-5 MI, 2-amino-3phosphonopropionicacid, and a-methylglutamic acid; and (2)a low-affinity ( L A ) system which transports aspartate (K, = 8 x lo4 M )and glutamate (K, = 1.2 x M )and is inhibited by glutamine. 24

Amino Acids and Paptides Linkage


Transport Defect in Mutants however, this conclusion must remain tentative-particularly in view of the large number of aromatic amino acid transport systems known t o be present in other bacterial species (see E. coli. S. typhimurium).

Slow growth on arginine as carbon source (Kay and Gronlund, 1969~).

Defective uptake of arginine (Kay and Gronlund. 1969aJ.

Slow growth on isoleucine as carbon source (Kay and Gronlund, 1 9 6 9 ~ ) .

Defective uptake of isoleucine (Kay end Gronlund, 1969e).

Resistance t o 4-mathyltryptophan [Hechtman and Scriver, 1970a.b).

Greatly reduced uptake of p-alanina and partially reduced uptake of Lalanine and proline; attributed t o e defect in the common transport system for these three amino acids (Hechtman and Scriver, 1970a). A t low temperatures the mutant did show facilitated diffusion o f p-alanine. suggesting that the block may be in energy coupling rather than In the carrier (Hechtman and Scriver, 1970b).

Resistance t o h y d r o x y - l lysine (Friede etel., 1972).

The hydroxylysine-resistant straln was found t o take up lysine more slowly than the wild type, end is believed t o be defective in the specific lysine transport system. The results are complicated by the fact that the double reciprocal p l o t of lysine uptake is still curvilinear i n the mutant, however. and a detailed kinetic analysis has not yet bean made (Friede e t a/., 19721.

Inability t o use arginine as an energy source (Barter and Strsughn, 1971).

Defective in the uptake o f arginine and citrulline (measured indirectly) (Baxter and Straughn. 1971).

Penicillin treatment in lowglutamete medium (Utech etal., 1970).

Mutant strain R4 lacks the H A system for aspartate end glutamate. The L A system remains, with Km's of 8 x lo4 M for aspartate and 9.6 x 10-3 M for glutamate (Utech eta!., 1970).

(Continued)


Transport System

Specificity

Streptococcus strain Challis

Alanine, glycine

Wild-type S. faecah (Mora and Snell, 1963) and wildtype Streptococcus strain Challis (Reitz et a/., 1967) contain a transport system for glycine and D- and L-alanine, inhibited by D-cycloserine.

-

Neurospora crassa

Neutral L-amino acids

Transport system I (Pall, 1969). present i n rapidly growing cells, takes up most neutral L-amino acids (DeEusk and DeEusk, 1965; Stadler, 1966; Lester, 1966; Wiley and Matchett, 1966; Pall, 1969; Wolfinbarger and DeEusk, 1971a). Km's have been reported for leucine (1 .2 x 104 M I ,valine (4.7 x 1 0 4 M I ,tryptophan (5-6 x 10-5 M ) ,phenylalanine (5-6 x 10-5 M ) , and histidine (6.5-8 x 1 0 4 M ) (Wiley and Matchett, 1966,1968; Pall, 1969; Pall and Kelly, 1971; Magill eta/., 1972). System I also takes up acidicamino acids (en.. L-aspartate) a t low p H (Wolfinbarger eta/.,

mtr

1971). Recently, an amino acid-binding protein has been isolated from the osmotic shock fluid of early exponentialphase Neurospora hyphae, and has been postulated t o play a role in transport system I (Wiley, 1970). The protein binds tryptophan with a K D of 8x M, very similar t o the K, for tryptophan transport; tryptophan binding is inhibited by other neutral amino acids (phenylalanine, leucine) but not b y arginine or lysine; and both the binding protein and the activity of the transport system are repressed b y growth in the presence of tryptophan, and are decreased in at least onemtr mutant (Wiley, 1970).

A glycoprotein, extracted from Neurospora conidia b y KCI, has also been postulated t o be a component of system I; it is absent in pmn mutants (but also in pmb mutants; see below) (Stuart and DeBusk, 1971) The relationship between this glycoprotein and the neutral amino acid-binding protein has not been discussed.

26

Gene


Linkage group I V (Stadler, 1966). The mtr locus has been extensively studied, since techniques are available t o select for b o t h forward mutations ( b y means o f analog resistance) and reverse mutations ( b y the ability of an amino acid auxotroph to grow at l o w amino acid concentrations) (Stadler, 1967; Brink eta/., 1969). Finestruc ture mapping has revealed three clusters o f mutational sites; no intragenic complementation was observed (Stadler and Kariya, 1969). mfr- is recessive t o the wild-type allele (Stadler, 1966).



Resistance to D-cycloserine. In addition t o transport m u tants, other D-cycloserineresistant mutants were f o u n d that contained increased amounts o f D-alanine: Dalanine ligase and alanine racemase, enzymes k n o w n t o be inhibited b y D-cyclow i n e (Reitz e f a/., 1967).

Defective in the uptake o f D- and Lalanine (glycine uptake n o t measured) (Reitzef a/., 1967).

Resistance t o 4-methyltryptophan (Lester, 1966; Stadler, 1966) orp-fluorophenylalanine (Stadler, 1966).Sixty independently isolated mutants, resistant t o b o t h analogs, have all been shown t o map i n t h e same region o f linkage group I V (Stadler, 1966; Stadler and Kariya, 1969);onlyone mutant has been isolated w i t h this pattern o f resistance and found t o be unlinked t o mfr (Stadler and Kariya, 1969).

Defective in system I . m f r mutants were originally f o u n d t o have decreased ( b u t s t i l l measurable) transport rates f o r neutral amino acids, including tryptophan, phenylalanine, tyrosine, methionine, valine, leucine, and histidine (Lester, 1966; Stadler, 1966). Subsequently, it was shown that the remaining uptake o f neutral amino acids (tryptophan, leucine) i n at least one mfr mutant was c o m pletely inhibited b y arginine, indicating that it occurred via system II (see below); b y contrast, neutral amino acid uptake in wild-type Neurospora is only partially inhibited b y arginine and involves b o t h systems I and II (Pall, 1969). Several rnfr mutants have been described that may have qualitative alterations in system I. These include tryp-auxotrophs which have become resistant to inhibit i o n b y phenylalanine (Stadler, 1966); a temperaturesensitive mfr mutant (Stadler and Kariya, 1969);a mutant w i t h decreased affinities f o r neutral amino acids (Pall, personal communication); and n e d mutants (see below). None o f these strains has been studied in detail, b u t qualitatively altered mtr mutants should prove useful i n establishing whether mtr is a structural gene for transport system I, and also in testing the hvpothesis that the neutral amino acidbinding protein (Wiley, 1970) and the KClextractable glycoprotein (Stuart and DeBusk, 1971) are involved in transport.

27

(Continued)

TABLE I Mutations Affecting Membrane Transport (Continued) ~

~~

~~

Amino Acids and Peptides arganism

Transport System

Specificity

Gene

Neurospora crassa IC0n~t.1

Most amino acids

Transport systam II (Pall, 1969) i s active in 3day hyphae. and takes up a wide variety of amino acids: D and L, 0: and fl, neutral, basic, and acidic, Km's have been measured fo[ L-tryptophan (45x 1 0 6 Mi, L-phenylalanine (2 x 10-6M ) , 0-phenylalanine (2-3 x 106 M I . glycine (5 x 10-6MI. L-aspartate (12 x 104 M ) . and L-asparagine (8 x 10-6 M ) ;and Ki's have been measured for several additional amino acids (Pall. 1969, 1970al.The activiry of system I I increases when cells are starved for carbon or nitrogen (Pall, 1969; SanchezataL. 1972).

su-mtr

'The symbols for Neurospora genes have been modified, where necessary, to follow the recommendations of Barratt and Perkins (1965) on Neurospora genetic nomenclature.

28




Linkage group I V ; probably allelic w i t h mtr (Wolfinbarger and DeBusk, 1971a).

Resistance t o p-fluorophenylalanine (Wolfinbarger and DeBusk, 1971a).

Defective in the transport of phenylbut not argialanine (via system I?) nine (Wolfinbarger and DeBusk, 1971a).

Has not been mapped;

Inability of a his- auxotroph t o grow o n histidine in the presence o f arginine, which blocks systems II and Ill (the other routes by which histidine could be taken up; see below) (Woodward e t a/., 1967; Magill era/., 1972).

Defective in system I. Residual uptake of histidine b y t h e neua mutant was completely blocked b y arginine. Systems I I and I l l were shown t o be present, with normal Km's and Vmax's; and histidine.once taken up, was shown t o be incorporated normally into protein (Magill etal., 1972).

Ability of a his-auxotroph t o grow on histidine in the presence of methionine and arginine, which block systems I , Il,and Ill,thereby inhibiting the growth of the parent strain (Magill e t a/., 1972).

Believed to be altered in system I , Histidine uptake b y system I i n the neur mutant required unusually high concentrations of methionine for inhibition (20 m M instead of 2 mM). suggesting that the relative affinities of system I for the various neutral amino acids may have been altered; this idea has not yet been checked directly.

it will be important t o

establish whether neua is allelic t o mtr (and neur; see below),

Linkage group IV (Magill eta/., 1972); may be allelic t o mtr.

Systems II and III were shown t o be present in neur; and histidine, once taken up, was incorporated normally into protein (Magill eta/., 1972). Linkage group I (Stadler, 1967; Brink etal., 1969).

Ability of a t r y p - m t r parent strain to grow on low concentrations of tryptophan (Stadler, 19671,or ability of a his-mtr strain t o grow on histidine in the presence of arginine (which would normally block the uptake of histidine via systems 1 1 and Ill;see below) (Brinketa/., 1969). Of 109 mutants isolated b y the latter method.49 proved to b e mtr+ revertants and 60 t o b e s u m t r (Brink eta/., 1969). Allsu-mtr isolates mapped in the same region of linkage group I; furthermore, the suppressors were not ailelespecific, and any su-mtr could suppress any mtr (Stadler. 1967; Brinketal., 1969).

29

Believed t o be regulatory mutants with increased levels of system I I (Pall, 1968). This explanation is consistent with t w o observed properties of su-mtr strains (Stadler, 1967): (1) they have recovered the ability t o take up neutral amino acids (tyrosine, leucine, isoleucine. valine, methionine, cysteine), but uptake has become sensitive t o inhibition by arginine and lysine; and (2) they have regained sensitivity t o p-fluorophenylalanine but are s t i l l resistant t o 4-methyltryptophen. This latter finding, unexplained at the t i m e s u m t r strains were originally described, is consistent with the known properties of system II, which has a 20-fold lower affinity for tryptophan (and possibly for tryptophan analogs) than for phenylalanine (Pall, 1969).

(Continued)

TABLE I Mutations Affecting Membrane Transport (Continued) A m i n o Acids and Peptides Organism

Transport System

Specificity

Gene

Neurospora crassa (Con%)

Basic L-amino acids

Transport system Ill (Pall, 1970a) i s present in rapidly growing cells, and takes u p basic amino acids (Bauerle and Garner, 1964; Roess and DeBusk, 1968; Pall, 1970a; Wolfinbarger and DeBusk, 1971a). Km's have been reported for L-lysine (K, = 5 x 1 0 - 6 M ) . L-arginine (K, = 2.3 x 1 0 - 6 M ) , a n d L-histidine (K, = i.ex10-3~).

baf

bm-1

30



Transport Defect in Mutants su-mtr mutants may also have an i n creased level o f transport system I V (see below),and the possibility must be considered that this gene affects transport in an indirect way, b y altering carbon and/or nitrogen metabolism (Pall, personal communication).

Maps o n linkage group IV, about 2 5 recombination units distal t o cot (Thwaites, personal communication).

Isolated b y an indirect p r o cedure, making use o f t h e fact that the pyr-3arg-12S parent strain i s inhibited b y arginine; bat mutants are selected b y their resistance to arginine (Thwaites, 1 9 6 7 ) .

Defective i n system Ill, w i t h essentially normal activities o f systems I and II. The result is that arginine and lysine can still be taken u p b y bat, via system I I , b u t their uptake is i n hibited b y a wide range o f amino acids (Thwaites and Pendyala, 1969; Pall, 1970a).

Linkage group V (Wolf inbarger and DeBusk, 1971a).

Resistance to canavanine (Roess and DeBusk, 1968; Wolfinbarger and DeBusk, 1971a).

Defective in arginine and lysine u p take and (partially) in histidine uptake (via system I l l ? ) (Roess and DeBusk, 1968; Wolfinbarger and DeBusk, 1971a).

Linkage group II (Magill eta/., 1972).

Inability o f a his-auxotroph to grow on histidine in the presence o f methionine, which blocks systems I and II (Magill eta!., 1972).

Defective in system Ill?Uptake o f histidine b y the basa mutant was completely inhibited b y methionine Systems I and II were shown to be present, w i t h normal Km's and Vmax's, and histidine, once taken up, was shown t o be incorporated normally i n t o protein (Magill era/., 1972).

Resistance t o canavanine and thialysine (Sanchez et a/., 1972).

Defective in system I l l ? Uptake o f basic amino acids is reduced in bm-1 mutants and is completely inhibited b y neutral amino acids (Sanchez eta/., 1972).

Linkage group V (Woodward eta/., 1967).

Inability o f a his-auxotroph t o grow o n histidine in the presence o f a neutral amino acid (Woodward eta/., 1967).

Defective in system Ill?Reported t o have abnormally low amounts o f "basic amino acid-inhibited" histidine uptake (Woodward eta/., 1967).

Linkage group V I I (Choke, 1969).

A b i l i t y o f a his-auxotroph t o grow o n histidinol (Choke,

Possibly defective in basic amino acid transport (via system Ill?). Very indirect evidence based o n growth experiments alone: t h e growth o f hishlp-1 double mutants on histidine

1969).

31

(Continued)

TABLE I Mutations Affecting Membrane Transport Kontinoedl Amino Acids and Peptides Organism

Transport System

Specificity

Gene

Neurospora crassa (Con't.1

Acidic D-and L-amino acids (aspartate, glutamate)

Transport system IV (Pall, 1970b) is present in nitrogen-or sulfur-starved hyphae, and takes up L-aspartate (K,,, = 1.2 x lo6 M I , D-aspartate (K,,, = 5.4 x 10-6M),L-glutamate (K,,, = 1.6 x 106 M I , D-glutamate (K,,, = 9.0 x 10-5 M ) ,and L-cysteic acid (K, = 7 x 10sM).

The following two mutants may have defects in amino acid transport, but have not been well characterized.

-

hlp-2

32



Transport Defect in Mutants was completely inhibited by neutral amino acids, while the growth o f the his-parent strain required both a neutral and a basic amino acid for complete inhibition (Choke, 1969). Choke has suggested that the basic amino acid transport system may have been altered in h/p-1 to acquire an affinity for histidinol. Note: The relationship among the various genes affecting basic amino acid transport is not yet clear, particularly since transport experiments b y different investigators have been done under very different conditions. A careful kinetic study i s needed t o check on the possibility that there may be more than one basic amino acid transport system in Neurospora, and qualitatively altered mutants are needed to establish which of the genes are structural genes.

N o clear-cut system-lV mutants have been isolated.

Unlinked t o m t r (D. Boone, cited in Stadler and Kariya, 1969).

Spontaneous mtr-like mutant in h p A m t r 119 stock, with partial resistance t o 4 m e t h y l tryptophan and p-fluorophenylalanine. I n a his-background prevents growth on histidine in the presence of arginine (D. Boone, cited in Stadler and Kariya, 1969).

Not characterized.

Linkage group V I I (Choke. 1969).

Ability of a his-auxotroph to grow on histidinol (Choke, 1969).

N o t characterized directly. The growth of a his-hlp-2 double mutant on histidine was stimulated b y aromatic amino acids and inhibited b y methionine, isoleucine, valine, or asparagine. Choke 11969) suggested that hlp-2 might have an altered permease which had acquired the ability t o take u p histidinol, but no direct uptake measurements have been made.

33

(Continued)


Transport System

Neorospofa crassa

The remaining mutants show reduced uptake of one or more groups of amino acids, but are difficult to interpret in terms of known transport systems. They may represent regulatory genes or other genes that affect transport indirectly.

/Con’C.I

Specificity

Gene fpr- 1

mod-5

nap

on(55701t)

34

A m i n o Acids and Peptides Linkage Linkage group V (Kinsey and Stadler. 1969).

Linkage group V I (St. Lawrence era/., 1964).



Resistance o f an mtrsu-mfr parent strain (see above) t o p-fluorophenylalanine (Stadler, 1966; Kinsey, 1967; Kinsey and Stadler. 1969). fpr-1 i s also resistant t o 4 m e t h y l t r y p t o p h a n (Kinsey and Stadler, 1969).

N o t characterized in detail. Partial defect in the uptake o f phenylalanine, p-fluorophenylalanine, tryptophan, and leucine; normal uptake o f lysine (Kinsey and Stadler, 1969). Possibly a regulatory gene, since Pall (personal communication) f o u n d fpr-1 t o have reduced levels o f b o t h systems I and II.

Reverses the inhibition o f the

N o t characterized in detail. m o d 4 shows a decreased lag i n the uptake o f aromatic amino acids and dipeptides (St. Lawrence etal., 1964).

f r y p - 3 parent strain b y leucine, yeast extract, or peptone (St. Lawrence etal., 1964).

Linkage group V (Jacobson and Metzenberg, 1968).

Resistance t o ethionine and p - f luorophenylalanine. Later found t o be resistant t o aminopterin, glycine, and 4 m e t h y l tryptophan (Jacobson and Metzenberg, 1968).

Primary defect not clear. Very slow uptake of neutral and acidic amino acids (methionine, ethionine, phenyla1anine.p-fluorophenylalanine, a-aminoisobutyrate, aspartate, and glutamate); partially reduced uptake o f proline and lysine; normal uptake o f arginine, glucose, and sulfate. Normal oxygen consumption and growth rate (Jacobson and Metzenberg, 1968).

Linkage group V I (Davis and Zimmerman, 1965).

In an arg-parent strain, prevented the utilization o f arginine f r o m the medium (Davis and Zimmerman, 1965).

Primary defect n o t understood. Reduced uptake o f arginine, lysine, other unrelated amino acids, and uridine (but at least in the case o f arginine, uptake is abnormal only in NHq+-containing medium). Resistant t o p - f l u o rophenylalanine (Davis and Zimmerman. 1965). Primary defect n o t clear. Jacobson and Metzenberg (1968) reported that, like nap.55701 showed decreased uptake o f neutral and acidic amino acids (methionine, ethionine, phenylalanine,p-fluorophenylalanine, aspartate, glutamate, and possibly proline) b u t normal uptake o f basic amino acids (arginine, Iysine), glucose, and sulfate, and normal oxygen consumption and growth rate, Protoplasts o f 55701 appeared t o show increased osmotic fragility, and Kappy and Metzenberg (1967) suggested that the primary defect might be in some structural element o f t h e plasma membrane.

Linkage group I (Kappv and Metzenberg, 1967; Jacobson and Metzenberg, 1968) b u t see below.

35

(Continued)


Transport System

Specificity

Gene

Neurospora crassa (Con?.)

Saccharomyces cerevisiae

Basic amino acids (arginine, lysine)

There are two transport systems for the basic amino acids in S. cerevisiae: (1) one specific for L-lysine (K, = 2.5 x 1 0 6 M),not inhibited significantly by ornithine or arginine; and

36

lys-pl




Resistance t o citrulline, in a pyr-3arg-125 parent strain sensitive t o arginine and citrulline b y virtue o f feedback control of the accumulation of argininespecific carbamyl phosphate (Thwaites et a/., 1970). These mutants appear t o be allelic w i t h 55701 ,and a detailed complementation map of the locus-involving 80 independently isolated mutants-has been worked out (Thwaites e t a / . , 1970). More recently, a question has arisen concerning the link age relationships of the original im(55701t) strain Although Metzenberg (personal communication) found that the transport defect and the temperature sensitivity segregated together through 12 crosses, and that both are closely linked to mating type on Iinkage group I, Tisdale and DeBusk (1970) have reported that a "transport regulatory" gene segregates from the temperature4ens.itive gene, and is located about 20 map units from rryp-1 on linkage group I l l (DeBusk, personal communication) These differences remain t o be worked out Resistance to thiosine (Grenson, 19661.

Defective i n the specific lysine transport system (the high-affinity component of lysine transport), with the Vmax reduced in mutant strain R A 309 by a factor o f 50 (Grenson, 1966).

37

(Con tinued)

TABLE I Mutations Affecting Membrane Transport (Conrinued) Amino Acids and Peptides Organism

Transport System

Saccharornyces cerevisiae (Con %)

Specificity (2) one that transports L-arginine (Krn = 10-5 M )and,

Gene arg-pl

a t higher concentrations, L-lysine (Krn = 2.5 x 104 M )

and L a n i t h i n e (Km = 3 x lO-3M). Competitlvely inhibited by L-canavanine ( K ; = 6 x 10-5 M )and D-arginine (K; = 7.5 x l o 4 M ) (Grenson eta/., 1966).

Dicarboxylic amino acids (aspartate, glutamate, a-aminoadipate)

Not characterized in detail

Histidine

High-affinity transport system for L-histidine. Not inhibited by other naturally occurring L-amino acids, but weakly inhibited by 1-methylhistidine (Crabeel and Grenson. 1970).

Methionine

High-affinity transport system for L-methionine (K, = 1.2 x 10-6M); competitively inhibited by L-ethionine, DLselenomethionine, D-methionine (Gits and Grenson, 1967).

Most neutral and basic amino acids

A general transport system for a wide range of amino acids. K,'s have been reported for L-arginine (7.6 x 10-6 M),L-lysine (3.1 x MI, L-citrulline (7.4-8 x 10-5 M),and L-tryptophan (0.9-1.3 x MI. The general transport system also takes up L-histidine, Lserine, L-alanine, L-methionine. L-valine, and possibly L-glutamate, but not proline. Inhibited by ammonium ions (Grenson eta/., 1970).

(60615)

38

Amino Acids and Peptides Linkage Unlinked t o lys-pl (Grenson, 1966).

Method o f lsolatina Mutants

TransDort Defect in Mutants

Resistance to canavanine (Grenson eta/., 1966; BBchet ef a/., 1970).

Defective in the arginine transport system (which is also responsible for the l o w a f f i n i t y component of lysine transport), w i t h the Vmax reduced in mutant strain MG 168 b y a factor o f 2 6 (Grensonetal., 1966).

Inability of a lys-auxotroph to use ol-aminoadipate as a source of lysine (Joiris and Grenson, 1969).

Defective in the transport of aspartate, glutamate, and olaminoadipate, w i t h the Vmax reduced by a factor o f 100 in mutant strain MG 9 5 6 (Joiris and Grenson, 1969).

Slow growth of a his-auxotroph at low histidine concentrations (Crabeel and Grenson, 1970).

Defective in the transport o f histidine (Crabeel and Grenson, 1970).

Unlinked t o lys-pi or arg-pl (Gits and Grenson, 1967).

Resistance to low concentrations of L-ethionine (Gitsand Grenson, 1967).

Not linked t o any of the genes determining specific amino acid transport systems (see above; Grenson eral., 1970).

Resistance to canavanine i n an arg-pi parent strain, growing in the absence o f ammonium ions (so that the general transport system is functional; see Specificity) (Grenson era/., 1970). gap alone does not confer resistance to amino acid analogs, which can also be taken up by the appropriate specific amino acid transport systems; gap arg-pi double mutants are resistant to canavanine because they lack both the general and the argininespecific systems. gap mutants can also be recognized by their slow growth on citrulline, which appears t o be taken u p only by the general amino acid transport system (Grenson etal., 1970).

Defective in the general amino acid transport system. gap mutants can take u p most amino acids via the various specific transport systems, but they d o n o t exhibit the usual increase in uptake rate (signifying the appearance of the general system) when ammonium ions are removed from the medium (Grenson etal., 1970).

A wild-type strain derived from six species of Saccharornyces (Bussey and Umbarger, 1970b).

Strain 60615 has a decreased affinity for leucine transport compared with 1.05 x wild-type strain L P (K,'sof 10-3M and 3.0 x M, respectively).

39

Defective in the transport of methio nine (Gits and Grenson, 1967).

(Continued)


Transport System

Specificity

Gene

Saccharo myces cerevisiae

1Con't.l

60615/f 12

a@ (apf)

Aspergillus nidulans

7

N o t characterized.

fpa D



Transport Defect in Mutants Leucine-binding proteins have been isolated from both strains, and because their affinities differ by a similar factor ( K 's of 1.68 x 1 0 5 M and 2.47 x 10- M),Bussey and Umbarger (1970a)

9

have suggested that the binding protein may be involved i n transport. This appears t o be a transport system for a rather wide range of amino acids (leucine, isoleucine, valine, threonine, tryptophan, tyrosine; Eussev and Umbargar. 1970a.b). b u t its specificity has not been investigated in detail, and its relationship to the systems described by Grenson and coworkers has not been established. Comparisons are made even more difficult by the complex origin o f strain 60615 (see Method of isolating mutants).

Unlinked t o (Grenson er el., 1970).

-

Isolated from strain 60615 (see above) by resistance t o trifluoroleucine (Bussey and Umbarger, 1970b).

Derepressed for leucine (and valine and tyrosine! transport, w i t h no change in Km (Bussey and Umbarger. 1970a.b).

Resistan- t o ethionine (Sorsoli eta/., 1964; Surdin et at., 1965; Cherest and de RobichonSzu lmajster. 1966; de Robichon-Szulmajster and Chersst. 19661 or top-fluorophenylalanine (Grens.cn and Hennaut, 1971). aap mutants are also resistant t o canavanine, @-2-thienylalanine,thiosine, norleucine, aretidine-2carboxylic acid, cyclolaucine, and cycloserine (Grenson and Hennaut, 19711. They can be selected most effciently b y resistance to a pair of amino acid analogs (e.g., canavanine plus 0-thienylalanine) or by the ability t o use glycine as sole carbon source (Grenson and Hennaut, 1971). Resistance t o p-fluorophenylalanine. @aD mutants are also resistant t o ethionine and t o

Reduced activity of several amino acid transport systems (arginine. lysina. leucine, proline, general). In each case the Vmax is depressed but transport is not eliminated completely. Granson and Hennaut (1971) suggest t h a t aap (apf) may determine a factor common to the various amino acid transport systems.

41

The uptake of purines and pyrimidines is unaffected (Grenson and Hennaut, 1971).

Defective i n the uptake o f phenylalanine, tyrasine, tryptophan, methionine. leucine, and aspartic acid, and partially

(Conrinued)


Transport System

Gene

Specificity

~~

Aspergi/lus nidulans (Con'r.) Ochromonas danica

Methionine. wine

Not characterized.

Organism

Transport System

Man

Cystine, lysine, Kidney: In vivo clearance studies and in vitro transornithine arginine port experiments with kidney slices have pointed (kidney, intestine) t o the existence o f at least t w o (and probably three) transport systems for cystine and the dibasic amino acids in the kidney:

Specificity

Disease Cystinuria

( 1 ) a specific system for cystine, n o t inhibited by lysine, ornithine, or arginine (Fox eta/., 19641; ( 2 ) a system for lysine, ornithine, and arginine, not inhibited by cystine (Foxeta/., 1964);and

( 3 ) (less conclusively) a shared system for all four amino acids, postulated on the basis o f combined genetic and clearance data (Dent and Rose, 1951). However, attempts t o demonstrate in vitro a mutual competitive inhibition among the four amino acidsas would be expected for a shared system-have been unsuccessful (Fox e t a/., 1964). Rosenberget a/. (1967) were able t o detect t w o kinetically distinct lysine transport systems in both rat and human kidney slices, b u t which ( i f either) might correspond to the shared cystine-lysine-ornithine-arginine system was not established. Intestine: In the intestine the shared system has been demonstrated by oral loading experiments (Milne e t a/., 1961; Asatoor e t a/., 1962; Rosenberg e t a/., 1965) and by direct measurements o f transport in jejunal mucosa obtained by biopsy (Thier era/., 1964, 1965; McCarthy eta/., 1964). The specific cystine and lysine-ornithinearginine systems appear not t o be present in the intestine.

42

Amino Acids and Paptides Linkage

-

Method o f Isolating Mutants L-3-aminotyrosine and phenylanthranilic acid (Sinha, 1969).

defective in the uptake of histidine, glycine, and glutamic acid (Sinha, 1969).

Resistance to ethionine (Hochberg et al., 1972).

Decreased uptake o f methionine, ethionine, serine (Hochbergetal., 1972).

Mode of Inheritance Autosomal recessive (Harris et al., 1955a.b).

Phenotype

Urinary excretion of dibasic amino acids and cystine

Control Type 1 Type II Type I I I

Normal Markedly increased Markedly increased Markedly increased

I , l l , a n d I I I havebeen shown t o be multiple alleles o f the same gene, since I I I , II Ill, and I I II double heterozygotes all have cystinuric phenotypes (Rosenberg, 1966. 1967; Rosenberg etal., 1966b).

Transport Defect

Increased renal clearance of cystine, lysine, ornithine, and arginine (Dent and Rose, 1951; Dent e t al., 1954; Arrow and Westall, 1958; Robson and Rose, 1957; Doolan et al., 1957; Frimpter e t a/., 1962; Crawhall etal., 1969; Rosenberg and Scriver, 1969).

More recently, cystinuria has been subdivided into three types on the basis of combined studies of urinary excretion and intestinal transport (Rosenberg e t al., 1966a):

Homozygotes


Heterozygotes -

Normal Increased Increased

Postulated to be a primary defect in the transport of cystine, lysine, orni thine, and arginine via the shared system (Dent and Rose, 1951). The defect has been demonstrated clearly in the intestine b y oral loading experiments (Milne etal., 1961; Asatoor e t al., 1962; Rosenberg et al., 1965) and by transport measurements on jejunal rnucosa obtained by biopsy (Thier etal., 1964, 1965; McCarthy etal., 1964).

In the kidney, in vivo clearance studies are consistent with such a defect (see Phenotype), but in vitro transport experiments have given a Intestinal transport o f dibasic amino acids and cystine somewhat confusing picture. Homozygotes Heterozygotes The postulated shared transPresent port system for Absent cystine, lysine ornithine. and Cystine-present arginine has not been detected Lysine-absen t Present in normal kidney slices (see Specificit v ) ; no clearcut abnormality in cystine transport was found in kidney slices from cystinuric patients (Fox e t al.. 1964); and both o f the lysine transport systems seen in normal subjects were shown t o be present in vitro in cystinuric patients, although the data were n o t sufficient t o conclude whether the kinetic parameters o f either

(Continued) 43


Transport System

Specificity

Disease

Man (Con ‘t.I

“Isolated” cystinuria

Dibasic aminoaciduria

Neutral amino acids (kidney. intestine)

There appear t o be multiple transport systems for new tral amino acids in the human kidney and intestine:

(1) A common system for neutral a-amino acids w i t h aliphatic or aromatic side chains. Such a system seems likely on the basis of the urinary excretion pattern i n Hartnup disease (see Transport defect), b u t direct evidence for i t s existence has n o t yet been reported.

44

Hartnup disease

A m i n o Acids and Peptides Mode o f Inheritance

Phenotype

Transport Defect system had been altered (Rosenberg eta/., 1967). A likely explanation f o r the discrepancy between the in vivo clearance data and t h e transport results is that a shared reabsorptive system does exist in the kidney (and isdefective in cystinuria), b u t that its kinetics are obscured b y simultaneous secretion o f cystine and the dibasic amino acids.

Probably recessive (data f r o m a single family) (Erodehl eta/., 1967).

Increased clearance o f cystine (Brodehl era/., 1967).

Postulated t o be defective in t h e renal transport of cystine via the specific system.

Recessive (reported in Finland; Perheentupa and Visakorpi, 1965; Kekoma'ki eta/., 1967).

Severe protein intolerance. Increased renal clearance o f lysine and arginine (ornithine not measured) (Perheentupa and Visakorpi, 1965; Kekoma'ki etal., 1967).

Dominant (reported in Canada; Whelan and Scriver, 1968).

Increased clearance o f dibasic amino acids (Whelan and Scriver, 1968).

Postulated t o be defective in the renal transport o f lysine, ornithine, and arginine (and possibly, in three o f the four cases, in intestinal transport as well). The genetic relationship among the three forms o f dibasic aminoaciduria is n o t y e t clear (Scriver and Hechtman, 1970).

Recessive (reported in Japan; Oyanagi etal., 1970).

Severe mental retardation, physical retardation, and increased renal clearance o f dibasic amino acids (Oyanagi etal., 1970).

Autosomal recessive (reviewed in Rosenberg and Scriver. 1969).

Increased excretion o f many neutral amino acids (see Transport defect); pellagralike skin rash and temporary cerebellar ataxia (Baron etal., 1956). thought t o reflect niacin deficiency resulting f r o m the malabsorption o f tryptophan (Milne, 1963).

Kidney: indirect evidence ( f r o m clearance data) f o r a defect in the renal transport o f many neutral amino acids (alanine, serine. threonine, asparagine, glutamine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, hlstidine, and citrulline) (Baron etal., 1956; Jepson, 1972). Intestine: indirect evidence f o r a defect in the intestinal absorption o f tryptophan (oral loading experiments; Milneetal., 1960) and other neutral amino acids (Scriver and Shaw, 1962; Scriver, 1965). In virro studies are needed to c o n f i r m that there is a primary defect in the

45

(Continued)


Transport System

Specificity

Disease

Man 1Con't.l

Methion i ne (intestine)

(2) A specific system for methionine, postulated on the basis of methionine malabsorption syndrome but not characterized directly.

Methionine malabsorption

Tryptophan (intestine)

(3)a specific system for tryptophan, postulated on the basis of tryptophan malabsorption syndrome; not characterized directly.

Tryptophan malabsorption

Glycine, proline, hydroxyproline (kidney, intestine)

Three transport systems have been postulated for glycine, proline, and hydroxyproline in the human kidney (Scriver, 1967, 1968; Scriver and Wilson, 1967): (1) a common system for the three amino acids, responsible for the reabsorption of about 40% of the filtered glycine and more than 90% of the filtered proline and hydroxyproline a t normal plasma concentrations (Rosenberg and Scriver, 1969). This system was first suggested by the finding that patients with elevated plasma proline concentrations (due to an inherited disorder of proline catabolism; Scriver e t a/., 1961; Schafer et a/., 19621 and also normal subjects, after proline infusion (Scriver et a/., 1964). excreted increased quantities of glycine and hydroxyproline. Direct measurements on several mammalian tissues in vitro (rat kidney slices, Wilson and Scriver, 1967; hamster intestine, Lin e t a / . , 1962; fetal rat bone, Finerman and Rosenberg, 1966; rabbit renal tubules, Hillman et a/., 1968; Hillman and Rosenberg, 1969, 1970) have revealed inhibition among the three amino acids, as expected for a common transport system, but have indicated that there are also

(2)a specific high-affinity system or perhaps two systems for glycine (and alanine), and 46

Iminoglycinuria

Amino Acids and Peptides Mode of Inheritance

Phenotype

Transport Defect transport of this group of amino acids, leading to the observed abnormalities in absorption b y the kidney and intestine.

Probably recessive (data from a single family; Hooft etal., 1968).

White hair; convulsions; mental retardation; attacks of hyperpnea; large amounts of methionine and a-hydroxybutyric acid (thought t o be a bacterial degradation product of methionine) in the feces (Smith and Strang, 1958; Hooft etal., 1965).

Postulated t o be a primary defect i n the intestinal absorption of methionine. Consistent with this view, oral loading of methionine produced diarrhea and an increase in fecal and urinary a-hydroxybutyric acid, while oral loading of six other amino acids failed t o produce these effects (Hooft etal., 1965). In vitro transport studies have not been performed.

Probably recessive (data from a single family; Drummond e t a/., 1964).

Recurrent febrile episodes; growth retardation; irritability; constipation; hypercalcemia; increased excretion of indoles (bacterial degradation products of tryptophan), resulting in a "blue diaper syndrome" (Drummond e t a/., 1964).

Postulated t o be a primary defect in the intestinal absorption of tryptophan. Oral loading of tryptophan caused an increase in fecal tryptophan and in the urinary excretion of indoles (Drummond e t a/., 1964). but in vitro transport experiments have not been done.

Autosomal recessive. There appear t o be at least three phenotypic types of iminoglycinuria: Intestinal absorption of glycine, proline, and hydroxyproline

Urinary excretion of glycine, proline, and hydroxyproline

(1) Described by

Homozygotes

Heterozygotes

Homozygotes

Heterozygotes

Increased

Normal

Defective

-

Defective

-

Normal

-

Tadaer a/. (1965); Morikawa et a/.(1966) ( 2 ) Described by

Increased

(3) Described by Scriver (1968); Rosenberg et a/. (1968)

Increas-d (glycine) Normal (proline, hydroxyproline)

Goodman ef a/. (19C7)

Increased

Increased (glycine) Normal (proline, hydroxyprol ine)

These types of iminoglycinuria may represent different mutant alleles at the same locus, since Scriver (1968) has described a patient with iminoglycinuria who had one hyperglycinuric pagent and one normal parent.

47

Thought t o be a primary defect in the transport of glycine, proline, and hydroxyproline via the common system.

(Continued)

TABLE I Mutations Affecting Membrane Transport (Conrinued) Amino Acids and Peptides Organism

Man (Con ‘t.I

Transport System

Specificity

(3) a specific high-affinity system f o r proline and hydroxyproline (and alanine). Recently, i t has been shown that the various systems are easily distinguishable in the developing rat kidney, the common system being present at b i r t h and the high-affinity proline and glycine systems appearing at 1 and 3 weeks after birth, respectively (Baerlocher e t a / . , 1970, 1971a.b).

48

Disease

Amino Acids and Peptides Mode of Inheritance

Phenotype

Transport Defect

More recently Greene et a/. 11973) have reported a new type of hyperglycinuria in two brothers who appear to have a qualitatively altered glycine-proline-hydroxyproline transport system (see Transport defect). Detailed study of the parents was not possible, so it was not clear whether the two patients were homzygous for a new type of mutation affecting the common transport system, or whether they were doubly heterozygous for two d i f ferent mutations. The genetic relationship of this new type of hyperglycinuria t o the previously described types of iminoglycinuria has not been established.

Kidney: The most frequent cases of iminoglycinuria appear t o involve quantitative reductions i n the common transport system without any obvious qualitative change. Renal clearance measurements have indicated that at least some homozygotes lack the common system entirely (Scriver, 1968; Rosenberg ef a/., 1968). They reabsorb about 60% of the filtered glycine at normal plasma glycine concentrations, but show no inhibition of glycine reabsorption when proline is infused intravenously; by contrast, normal subjects reabsorb more than 95% of the filtered glycine, and show a decrease t o 50% during proline infusion. (The proline-insensitive glycine reabsorption in both cases is thought t o represent the activity of other glycine transport systems.) Some (but not all) heterozygotes appear t o have intermediate levels of the common glycine-proline-hydroxyproline system, since they excrete increased quantities of glycine but not proline or hydroxyproline (Scriver, 1968; Rosenberg et a/., 1968). (This pattern is consistent with the measured affinities of the common system for the three amino acids in normal subjects: lower for glycine than for proline and hydroxyproline.) I n addition, when the plasma proline concentration is increased b y infusion in such heterozygotes, the maximum tubular reabsorption of proline can be shown t o be reduced t o about one-half the normal value (Scriver, 1968). By contrast with these apparent quantitative alterations, the new form of hyperglycinuria described by Greene e t a / . (1973) appears t o represent a qualitative change in t h e glycineproline-hydroxyproline transport system. Glycine reabsorption is reduced ( t o 73% of the filtered load) and n o t further inhibited by proline. suggesting that no glycine i s being transported

(Continued) 49


Transport System

Specificity

Disease

Man (Con ‘t.)

Carbohydrates Organism Escherichia coli

Transport System L-Arabinose

Specificity

Gene

There are two inducible transport systems for L-arabinose in E. coli, both under the control ofaraC, the regulatory gene for the L-arabinose operon (Schleif, 1969; Brown and Hogg, 1971,1972):

( 1 ) a l o w a f f i n i t y system (K,

=

araE

1.O x 1 0 4 M ) and

(2) a high-affinitysystem (K, = 8 . 3 x 10-6M). Both are competitively inhibited b y D-fucose, Dxylose, and p-methyl-L-arabinoside; in addition the high-affinity system is inhibited by D-galactose (Brown and Hogg, 1972). A protein that binds L-arabinose has been isolated from E. c o l i b y Hogg and Englesberg (1969) and b y Schleif (1969),and appears to be involved in the highaffinity transport system (Brown and Hogg. 1972). The protein is released from the cells b y osmotic shock, suggesting that it i s located in the periplasmic space between the cell wall and the cell membrane (Neu and Heppel, 1965). I t has a molecular weight of 32,000 t o

50

araF

A m i n o Acids and Peptides Mode o f -

Inheritance

Phenotype

Transport Defect b y the common system. A t the same time proline is reabsorbed w i t h a shift in concentration dependence (maximal reabsorption being reached o n l y at higher filtered loads), suggesting a change in the a f f i n i t y o f the transport system f o r proline. Intestine: I t seems clear that t h e comm o n glycine-proline-hydroxyproline system also exists in t h e intestine since, in oral loading experiments, some patients w i t h iminoglycinuria exhibit an intestinal defect in t h e transport of these three amino acids (Morikawa eta/., 1966; Goodman eta/., 1967). Other patients d o not, however (Scriver, 1968; Rosenberg eta/., 1968).and it is n o t k n o w n whether they possess a different mutational alteration in the common system or whether other intestinal transport systems obscure the defect.

Carbohydrates Linkage



Near thy; unlinked t o araDABC (Isaacson and Englesberg, 1964; Englesberg era/., 1965).

Lack the low-affinity L-arabinose The growth o f araA and araD strains (which lack L-arabinose transport system (Brown and Hogg, isomerase and L-ribuloseS1971,1972);contain normal amounts phosphate 4epimerase. reo f the L-arabinose-binding protein spectively) is inhibited b y L(Schleif, 1969; Hogg and Englesberg, arabinose, and in at least some 1969). araA andaraD strains,araE mutants can be selected b y resistance t o arabinose (Isaacson and Englesberg, 1964; Hogg and Englesberg, 1969; Schleif, 1969; Hogg, 1971 ) . (araC-mutants are also selected b y this procedure but can be recognized b y their pleiotropic negative phenotype; see below.)

Map location n o t known. Unlinked t o araE and araDABC

Lack the high-affinity L-arabinose Identified in an araE parent strain b y the inability t o metab- transport system; in addition, t h e L-arabinose-binding protein is either olize L-arabinose (on tetrazo51

(Contimed)

TABLE I Mutations Affecting Membrane Transport (Continued) Carbohydrates Organism

Transport System

Specificity

Gene

35,000,consists o f a single polypeptide chain,and binds 0.8-1 .O moles o f L-arabinose per mole w i t h a K x o f 2-5.7 x 10-6 M (Schleif, 1969; Hogg and Englesberg, 1969). Binding, like transport via the high-affinity system, is strongly inhibited b y D-galactose (Brown and Hogg, 1972).

Escherichia coli (Con?.)

araC

A t least four d i f ferent transport systems exist in E. coli for galactose and galactosides: Lactose (TMG I )

An inducible system which transports w a n d p-D-galactosides (including lactose), p-D-thiogalactosides, and galactose. First described b y Cohen and Rickenberg (1955). Rickenberg e t a / . (1956),and Pardee (1957). K,'s for commonly used substrates are: lactose, 6-9 x 1 0 4 M; melibiose,2 x 1 0 4 M;o-nitrophenylQ-Dgalactoside (ONPG), 3-10 x l o 4 M; methyl-1-thio-p-Dgalactoside (TMG), 5 x l o 4 M; p-Dgalactosyl-1 -thiop-Dgalactoside (TDG),2-5 x 1 0 6 M; phenyl-1 -thiop-D-galactoside (TPG), 2.5 x 1 0 4 M (Kepes, 1960; Kepes and Cohen, 1962; Winkler and Wilson, 1966; Carter e t a / . , 19681.

52

lacy

~

~

~~




(Hogg and Englesberg, 1969;Schleif, 1969; Brown and Hogg, 1972).

liumarabinose agar medium) (Brown and Hogg, 1972). Earlier Hogg and Englesberg (1969) and Schleif (1969). b y means o f direct testing, had found t w o mutants w i t h decreased L-arabinose-binding activity. In addition, Hogg (1971) has described a method to screen systematically for binding mutants b y plating o n medium containing antiserum to t h e binding protein; mutants lacking the protein d o n o t produce a visible precipitin reaction.

altered (detectable as cross-reacting material) o r missing altogether (Brown and Hogg. 1972).

Between thr and leu. closely linked t o araD, araA, and araB tn t h e order DABC (Isaacson and Englesberg, 1964; Englesberg eta/., 1965).

araC-mutants can be isolated (along w i t h araA, araB, and araD mutants) b y their i n ability t o grow o n Larabtnose as sole carbon source (Englesberg, 1961 1. Alternatively, they can be selected in an araA o r a r a D genetic background b y resistance t o arabinose (see above; Hogg and Englesberg, 1969; Schleif, 1969).

A regulatory gene which exerts positive control over the operon araD araA JraB (coding f o r L-ribulose4phosphate 4epimerase, Larabinose isomerase, and L-ribulokinase, respectively) and also over t h e unlinked genesaraE and araF (thought t o code for components o f t h e low-affinity and h i g h a f f i n i t y arabinose transport systems; see above). araC- mutants are unable t o synthesize the three arabinose enzymes o r either o f the transport systems, while araCc m u tants synthesize these proteins constitutively (Englesberg eta/., 1964; Novotny and Englesberg, 1966; Englesberg eta/., 1965; Sheppard and Englesberg, 1967; Englesberg eta/., 1969; Brown and Hogg, 1 9 7 2 ) .

araCC mutants can be selected b y resistance t o D-fucose (Englesberg et a/., 1964; Doyle eta/., 1972). which normally inhibits induction o f t h e arabinose operon (Beverin eta/., 1971). Closely linked t o lac/ (the regulatory gene for the lactose operon), /acZ (the structural gene f o r pBalactosidase), and /acA (the structural gene f o r thiogalactoside transacetylase) in the order / Z Y A (Jacob and Monod, 1961 1 .

Inability t o ferment lactose (Rickenberg era/., 1956)./acY mutants are then distinguished f r o m l a c 2 (p-galactostdaseless) mutants b y t h e fact that they are cryptic; when treated w i t h toluene t o disrupt the cell membrane, they can hydrolyze ONPG. Resistance to O-nitrophenyl1-thio-p-Dgalactoside, which

53

l a c y is well documented to be t h e structural gene for t h e M protein (see Specificity), which in t u r n i s a c o m ponent o f the lactose transport system: M protein is not detectable in uninduced cells, indicating that i t s synthesis is under the regulation of the lac/ gene (see below), nor is it detectable in l a c y (amber) mutants (Foxeta/., 1967). One particular Y mutant (8204-ts3), studied in some detail, was shown t o have b o t h a tempera-

(Continued)


Transport System

Specificity

Gene

The kinetics of transport have been worked out in detail (reviewed b y Kepes, 197la,b),and have led t o t h e notion o f a saturable carrier which mediates either facilitated diffusion or, when coupled t o metabolic energy, active transport, Energy coupling in the latter case is viewed as having n o effect o n the entry o f substrate b u t instead as preventing exit, b y greatly reducing the affinity o f the carrier for substrate at the inner surface o f the membrane (Winkler and Wilson, 1966).

Escherichia (Con't.)

COIl

A membrane protein ( M protein) has been isolated f r o m cells that possess the P-galactoside transport system, b y taking advantage o f the fact that sulfhydryl groups o n the protein can be specifically protected f r o m reaction w i t h Nethylmaleimide if a tightly bound substrate (such as TDG; see above) is present (Fox and Kennedy, 1965; Fox eta/., 1967; Carter eta/., 1968). The protein has been solubilized f r o m t h e membrane b y means o f detergents; it i s a single polypeptide chain w i t h a molecular weight of 30,000 (Jones and Kennedy, 1969).

lac1

Melibiose ( T M G II)

A n inducible transport system f o r galactose and certain and pgalactosides, including melibiose (6-0-a-Dgalactosyl-Dglucose) , galactinol (6-O-a-D+alactosylD-glucitol), and TMG, b u t n o t lactose o r ONPG (Prestidgeand Pardee, 1965; Leder and Perry, 19671. Coordinately regulated w i t h the enzyme olgalactosidase (Buttin, 1968; Schmitt, 1968).

01-

54

melt3


Method of Isolating Mutants inhibits the growth o f cells that have a functional lactose transport system (Muller-Hill eta/., 1968; Wong er a/., 1970; T. H. Wilson eta/., 1970). Decreased ability t o accumulate TMG-14C as screened directly b y autoradiography o f colonies (Zwaig and Lin, 1966; Wong et a/., 1970; T . H. Wilson era/., 1970)

Transport Defect in Mutants ture-sensitive lactose transport system and a temperature-sensitive M protein ( F o x et a/., 1967). More recently,another class of rnutants ha: been isolated which appear t o possess qualitatively altered lactose transport systems (Wong eta/., 1970; T. H. Wilson eta/., 1970; Wilson and Kusch, 1 9 7 2 ) . These mutants contain a normal (or perhaps greater than normal) number o f lactose carriers, judged b y four assays that measure carrier activity independent o f coupling t o metabolic energy: (1) t h e rate of entry o f ONPG (facilitated diffusion; Winkler and Wilson, 1966); (2) the initial rate o f entry o f T M G ; (3) counterflow o f T M G ; and (4) direct assay o f the M protein. Active transport ( o f T M G ) is reduced drastically in the mutants, however, and the defect has been traced t o an abnormally high exit rate (which is not a generalized leak, since it is specific f o r substrates of t h e lactose transport system). One mutation o f this t y p e has been mapped in (or near) the lactose operon, consistent w i t h t h e idea that it is a mutation in the Y gene, causing a qualitative change in the M protein such t h a t transport is n o longer energy-coupled.

See above.

Variety o f methods reviewed b y Gilbert and Muller-Hill (1970).

Secondary changes in the level o f lactose transport and also in the levels o f P-galactosidase and thiogalactoside transacetylase; lac/ i s t h e regulatory gene of the lactose operon (Jacob and Monod, 1961).

Closely linked t o metA (Schmitt, 1968).

Inability t o grow o n melibiose; mutants lacking the transport system must then be distinguished ( b y direct assay) f r o m those lacking olgalactosidase and f r o m regulatory mutants deficient in both (Buttin, 1968;Schmitt. 1968).

Defective in the uptake o f melibiose and other galactosides (Schmitt, 1968; Buttin, 1968); n o t characterized in detail.

Considerable differences exist among the various wild-type strains of E. coli w i t h respect 55

(Continued)

TABLE I Mutations Affecting Membrane Transport (Continued) Carbohydrates

Organism

Transport System

Specificity

Gene

Escherichia coli (Con't.1

p-Methylgalactoside

A n inducible transport system for galactose and pgalactosides, with K,'s of 5 x 10-7 M for galactose (Rotman and Radojkovic. 1964); 2.8 x 10-6 M for 1 -09-D-galactosyl-glycerol (Boos, 1969); and 2 x 1 0 3 M for methyl-1 9-D-galactoside (MG; Rotman er a/., 1968). Also studied b y Rotman (1959). Horecker eta/. (1960a,b), Osborn etal. (19611, Buttin (1963a,b), Ganesan and Rotman 11966). Booset a/. (1967). Boos and Wallenfels (19681,and Singer and Englesberg (19711,This system, rather than the one described below, plays the key physiological role in growth on low concentrations of galactose and i n the induction of thegal operon (Wu and Kalckar, 1966; Wu, 1967; Wu eta/., 1969; Kalckar, 1971 1. A galactose-binding protein, which appears t o be involved i n transport via the 0-methylgalactoside system, was first described by Anraku (1968a,b,c), and has been characterized i n detail by Boos (1969), Boos and Gordon (1971),and Boosetal. (1972). Like the transport system, the binding protein has the highest affinities for galactose, P-galactosyl-glycerol, and glucose; and i n addition, the protein has been shown t o undergo a conformational change in the presence of substrate, detected as an increase in electrophoretic mobility and in fluorescence. Genetic evidence suggests that the binding protein also plays a role in galactose chemotaxis (Hazelbauer and Adler, 1971; Kalckar, 1971; Boos, 1971) (see Transport Defect i n Mutants).

56

mglP




t o the melibiose operon; i n duction o f the permease and a-galactosidase is temperatureSensitive in E. cot; K-12 and does n o t occur during growth at 37' or higher (Buttin. 1968); E. coli B is inducible at all temperatures; and E. c o l i ML lacks the permease and a-galactosidase altogether (Pardee, 1957; Prestidge and Pardee, 1 9 6 5 ) . One mutant (W4345) which lacks the p-methylgalactoside permease was shown t o be closely linked t o his (Ganesan and Rotman, 1966); t h e remaining mutants have not yet been mapped.

W3092i was isolated as an "inducible" mutant f r o m a galactokinaseless parent strain that is normally "constitutive" f o r the other t w o enzymes o f the gal operon (galactose-1 -phosphate uridyltransferase and UDPGal 4-epimerase); the p-methylgalactoside transp o r t system, when functional, maintains a high intracellular concentration of galactose in the absence o f the kinase, and the galactose in t u r n serves as an endogenous inducer o f the gal operon (Wu and Kalckar, 1 9 6 6 ) . More recently, p-methylgalactoside transport mutants have been isolated o n the basis o f lowered accumulation o f galactose-l4C b y an autoradiographic technique (Boos and Sarvas, 1970),or b y t h e inability t o show chemotaxis toward galactose (Hazelbauer and Adler. 1971).

57

Defective in the p-methylgalactoside transport system, w i t h little o r no u p take o f galactose (at l o w concentrations), p-methylgalactoside, o r galactosyl-glycerol (Wu, 1967; Wu eral., 1969; Boos and Wallenfels, 1968; Boos, 1969). It has not yet been established that this phenotype corresponds to a single gene, since o n l y one o f t h e mutants has been mapped (see Linkage). More is k n o w n about the biochemistry o f the mutants. Some (but not all) o f t h e m contain reduced levels o f galactosehinding protein (Boos and Sarvas, 19701,and recently t w o mutants have been identified w i t h qualitatively altered galactosehinding proteins: ( 1 1 Strain AW550 (isolated b y i t s failure t o show chemotaxis at l o w galactose concentrations and also found t o be defective in galactose transport; Hazelbauer and Adler, 1971 ). A p r o tein has been isolated f r o m this strain which cross-reacts w i t h antibody t o the wild-type galactose-binding protein b u t does not b i n d galactose at low concentrations o r show the substrate-induced conformational change characteristic of t h e wild-type protein (Boos, 1971). (2) Strain EH3039 (defective i n t h e p-methylgalactoside transport system). Protein f r o m this mutant does n o t b i n d galactose at l o w concentrations o r show a substrate-induced conformational change detectable b y acrylamide gel electrophoresis, b u t does-at high concen-

(Continued)

TABLE I Mutations Affecting Membrane Transport (Conrinued) Carbohydrates Organism

Transport System

Specificity

Gene


mglR

Galactose

A n inducible system which is specific f o r galactose (K, = 1 0 4 M ) (Rotman etal., 1968).

(galP)

Hexose phosphates

A n inducible system which transports a variety o f hexose phosphates, including glucose G-phosphate, mannose Gphosphate, fructose G-phosphate, 2 d e o x y glucose Gphosphate. glucose 1 -phosphate, and fructose 1-phosphate (Fraenkel etal., 1964; Pogell etal., 1966; Winkler, 1966; Dietz and Heppel, 1971a.c; Ferenci etal., 1971). The K, for glucose Gphosphate is 2 . 7 5 x 1 0 4 M,and Ki's for mannose 6-phosphate and fructose 6-phosphate are 5 x 1 0 4 M and 4 x l o 4 M, respectively (Pogell etal., 1966; Winkler. 1 9 6 6 ) . Interestingly, the system is induced b y exogenously added, b u t not b y endogenously formed, glucose Gphosphate (Heppel. 1969; Winkler, 1970,1971b; Dietz and Heppel, 1971b).

uhp



Transport Defect in Mutants t r a t i o n s 4 x h i b i t a substrate-induced increase in fluorescence. A t r y p t i c digest o f the mutant protein contained one altered peptide. A revertant-selected for recovery o f transport ability-contained a normal galactose-binding protein, as judged b y binding affinity, electrophoresis, fluorescence, and peptide mapping (Silhaw and Boos, 1972; Boos, 1972). These results indicate that strain EH3039 land probably strain AW550 as well) carry mutations i n the structural gene for the galactose-binding protein (although this gene has not yet been shown t o be mglP), and furthermore that the binding protein plays an obligatory role in b o t h the p-methylgalactoside transport system and galactose chemotaxis.

Maps between 5 6 a n d 74 minutes (Lengeler e t a/., 1971).

A b i l i t y o f carbonstarved cells t o grow o n p-methylgalactoside (Lengeleretal., 1971) .

mg/R mutant L104 is constitutive for p-methylgalactoside transport b u t s t i l l f u l l y inducible f o r the gal operon; mg/R i s concluded t o be a regulatory gene f o r m g l P (Lengeler eta/., 1971 1.

Selected b y penicillin treatment o f cells on a glucose G-phosphate medium (Winkler, 1966) or, starting w i t h a PEPcarboxylase-negative parent strain growing o n acetate as carbon source, b y resistance t o growth inhibition b y glucose 6-phosphate. (Kornberg and Smith, 1969). This latter metho d is based on the fact that d u r ing growth of PEP-carboxylasenegative cells o n acetate, C4 acids are synthesized b y t h e glyoxylate cycle; and isocttrate lyase, the key enzyme o f the glyoxylate cycle, i s inhibited b y C3 acids (PEP, pyruvate) derived f r o m glucose 6-phosphate.

Defective in the transport of hexose phosphates (Winkler, 1966; Kornberg and Smith, 1969).

N o t linked t o his (Ganesan and Rotman. 1966). Maps near pyrE (Kornberg and Smith, 1969).

59

(Continued)


Transport System

Specificity

Gene uhpc


L-aGlycerophosphate

A transport system, inducible b y L-a-glycerophosphate, conveniently studied in a mutant lacking alkaline phosphatase and LIU-GP dehydrogenase (so that L-a-GP can neither be hydrolyzed extracellularly nor metabolized intracellularly). Under these conditions LIU-GP IS accumulated, unchanged, in an energy-de, o f 1.2 x 1 0 6 M. Uptake pendent process w i t h a K is competitively inhibited b y high concentrations o f ~ DL-glyinorganic phosphate (Ki = 7.5 x 1 0 - 3 or ceraldehyde 3-phosphate (Ki = 5 x 1 0 4 M ) (Hayashi eta/., 1964).

g/P T

Glycerol

A system for the facilitated diffusion o f glycerol, induced by glycerol (in cells possessing glycerol kinase) or b y L-a-GP (Sanno eta/., 1 9 6 8 ) .

glpf

60

~

~~




Closely linked t o ohp (Ferenci and Kornberg, 1971).

Selected, in a wild-type strain, b y the ability t o grow on fructose 1-phosphate (which is a substrate f o r , b u t cannot induce,the hexose phosphate transport system) (Ferenci e t a / . , 1971) or, in an enzymeI mutant (see below), b y the ability t o grow o n glucose 1 -phosphate (Dietz and Heppel, 1 9 7 1 ~ )(In . the absence o f enzyme I o f the phosphotransferase system, glucose 1-phosphate can support growth o n l y if it can be taken u p b y the hexose phosphate transport system.)

F o r m the hexose phosphate transport system constitutive1y;ohp may be an operon, w i t h uhpc mutations in the control gene (Ferenci and Kornberg, 1971; D i e t z a n d Heppel, 1 9 7 1 ~ ) .

Maps at 42 minutes, between t y r and his (Cozzarelli era/., 1968) and close o r adjacent t o g/pA, the gene f o r the anaerobic L a - G P dehydrogenase (Kistler era/., 1969; Kistler and Lin, cited in Berman-Kurtz era/., 1971).

Isolated b y the inability t o ferment L a G P (Hayashi eta/., 1964). b y slow growth o n a dual carbon source consisting o f a large amount o f L a - G P and a trace of casein hydrolyzate (Cozzarelli e t a / . , 1968). or b y resistance to phosphonomycin, an antibiotic which is taken up b y the L a G P transport syst e m (Hendlin eta/., 1969).

Defective in L a G P transport. g / p T is believed t o be the structural gene for the transport system (rather than a regulatory gene) because all L a - G P transport mutants map in the same small region, well separated f r o m the k n o w n regulatory geneglpR (see below), and because residual transport in g / p T mutants is still inducible in the normal way (Cozzarelli etal., 1968). Defective in t h e facilitated diffusion of glycerol (Sanno, cited in EermanKurtzeral.. 1971).

Maps at 7 6 minutes, close o r adjacent t o g/pK, t h e gene f o r gly cerol kinase (Cozzarelli and Lin, 1966; Sanno, cited in Eerman-Kurtz et a/., 1971). Maps a t 66 minutes, close o r adjacent t o g/pD (the structural gene for the aerobic L a - G P dehydrogenase) b u t not near the other genes it regulates: g/pT, A,

Constitutive, noninducible. and temperaturesensitive control mutants have been isolated b y several methods, described in detail b y Cozzarelli e t a / . (1968).

f , K (Cozzarelli e r a / . . 1968; Berman-Kurtz etal., 1971).

Secondary alterations in the transport of L a - G P and the facilitated d i f f u sion of glycerol.g/pR is thought t o be a regulatory gene, exerting control over at least three operons: g l p T a n d g/pA ( L a - G P transport and anaerobic L a - G P dehydrogenase; Cozzarelli eta/., 1968; Kistler and Lin, cited in Berman-Kurtz et a/., 1971 );g/pF and g / p K (facilitated diffusion o f glycerol and glycerol kinase; Sanno, cited i n Berman-Kurtz eta/., 1971; Cozzarelli and Lin,

-

61

(Continued)


Transport System

Specificity

Gene


Ma1t ose

First described b y Wiesmeyer and Cohn (1960); kinetics have not been analyzed in detail.

ma16

malT

Phosphotransferase system

A system that carries o u t the phosphorylation of a variety o f sugars and their "group translocation" i n t o bacterial cells according t o the following scheme: enz I

Phosphoenolp y r uvat e

t

HPr

7Phospho-H Pr + pyruvate

Phospho-HPr

+

62

enz II sugar

Sugar-P + HPr

ptsl (ctr)



Transport Defect in Mutants 1966); and glpD (anaerobic L a - G P dehydrogenase; Cozzarelli eta/., 1968).

The m a l B region, located between m e t A and u v r A (Schwartz, 1966). appears t o include more than one gene involved in maltose transport (cited i n Thirion and Hofnung, 1972). I n addition, it includes a gene (/amB) required for the synthesis o f A bacteriophage receptors (Thirion and Hofnung, 1972). and polar mutants in malB are often b o t h defective in maltose transport and resistant t o A (Lederberg, 1955; Schwartz, 196713; Thirion and Hofnung, 1972).

Isolated, in E. coli strain K-12, b y the inability t o grow on maltose as sole carbon source; must be distinguished f r o m other rnaltose-nonutilizing mutants in t h e m a l A and m a l B gene clusters (Schwartz, 1966, 1967a,b; Hatfield etal., 1969). E. coli strain B,which is u n able t o grow on maltose and is resistant t o bacteriophage A , i s considered t o be ma/B(Chung and Greenberg, 1968; Ronen and Raanen-Ashkenazi, 1971).

Inability t o grow o n maltose as Located in the ma/A sole carbon source (f.coli region. together w i t h malP (thought t o be the K-12; see above). structural gene for maltodextrin phosphorylase) and malQ (thought t o be the structural gene for amylomaltase) (Hatfield etal., 1969; Hofnung and Schwartz, 1971 ; H o f nungetal., 1971 1. Map between purC and supN (Epstein etal., 1970); this location is consistent w i t h the data o f Wang e t a/. (1969) and Bourd eta/. (1968).

Inability t o grow o n glucose (strain MM-6; Monod, cited in Asensio eta/., 1963; Fraenkel etal., 1964; the ctr mutants of Wang and Morse, 1966,1968; Wangetal., 1969); inability t o ferment fructose + rnannose ( F o x a n d Wilson, 1968); inability t o ferment mannitol + sorbitol (Epstein etal., 1970).

63

Believed t o have a primary defect in the transport o f maltose, b u t n o t characterized in detail (Schwartz, 1966,1967abl.

Defective in maltose transport, a m y lomaltase, and maltodextrin phosphorylase, and resistant t o bacteriophage A.malT is thought t o be a regulatory gene exerting positive control over b o t h t h e A and B operons (Schwartz, 1967b; Hatfield etal.,

1969).

Defective in the uptake o f a variety o f sugars and in enzyme I. The m u tants can be divided i n t o t w o classes o n the basis o f their growth characteristics and the amount o f remaining enzyme I : ( 1 ) "Leaky" mutants, which have significant residual enzyme-I activity (Fox and Wilson, 1968; Saier eta/., 1970; Epstein etal., 1970). These strains d o n o t grow, o r grow slowly, o n sugars k n o w n t o be substrates for the phosphotransferase system in E. coli, including glucose, fructose,

(Continued)

TABLE I Mutations Affecting Membrane Transport (Continued) Carbohydrates Organism Escherichia c o l i (Con't.)

Transport System

Specificity

Gene

First described b y Kundigetal. (1964) and reviewed b y Roseman (1969). Enzyme I and the low-molecularweight protein, HPr, are common t o all sugars phosphorylated b y the system and are found primarily in the cytoplasm; enzyme I has been extensively purified f r o m E. c o l i IM. Saier, cited in Kundig and Roseman, 1971a). and HPr has been obtained in homogeneous f o r m f r o m E. coli (Anderson e t a / . , 1968) and S. typhimurium ( A . Nakazawa, cited in Kundig and Roseman, 1971a). By contrast there are a family of "enzymes 11,"

which

c a n be distinguished f r o m one another on the basis o f their specificities toward different carbohydrates. Each "enzyme 1 1 " i s a complex, membrane-bound fraction; recently,the constitutive enzymes II (for glucose, mannose,and fructose) of E. c o l i have been shown t o contain at least t w o proteins (11-A and Il-B),and t o require phosphatidylglycerol and a divalent cation (Ca2+ o r Mg2+) for activity (Kundig and Roseman, 1971b).

In E. coli and S. typhimuriurn, relatively few sugars are substrates for t h e phosphotransferase system, while In in S. aureus, all sugars that have been examined are substrates for the system (see below).

64



Transport Defect in Mutants mannose, mannitol, and sorbitol [Tanaka eta/., 1967; F o x and Wilson, 1968; Epstein eta/., 1970 (except that the latter strains d o grow o n glucosell.

(2) "Tight" mutants, which have very l o w levels o f enzyme I ( F o x and Wilson, 1968; Wangetal., 1969; Saier e t a/., 1970; Epstein eta/., 1970; Morseetal., 1971l.These strains grow poorly o n t h e above sugars and in addition o n other compounds (maltose, lactose, melibiose, glycerol, succinate) which are not substrates f o r the phosphotransferase system (PTS) in E. coli (Wang and Morse, 1966,1968; Fox and Wilson, 1968; Epstein eta/., 1970). It has been suggested that the failure of this second group of mutants t o grow o n non-PTS sugars stems f r o m an i n creased sensitivity t o catabolite repression, such that the enzymes required t o metabolize t h e sugars are n o t induced (Pastan and Perlman, 1969; Berman eta/., 1970; Epstein eta/., 1970). Consistent w i t h this view is the fact that cyclic AMP, added t o the medium, can restore b o t h inducibility and growth (Pastan and Perlman, 1969; de Crombrugghe era/., 1969; Berman eta/., 1970; Epstein eta/., 1970; Dahl etal., 1971). In addition,growth o n i n d i vidual non-PTS sugars can be restored b y suppressor mutations which i n crease the synthesis o r t h e activity o f the corresponding enzymes w i t h o u t affecting the level o f enzyme I (Wang and Morse, 1967; Morse e t a/., 1969; Wangetai., 1970; Bermaneta/., 1970; Berman and Lin, 1971: BermanKurtzetal., 1971; Dahl eta/., 19711. Recently, a temperature-sensitive pts/ mutant has been isolated and shown t o possess a heat-labile enzyme I; this result indicates t h a t p t s l i s the structural gene f o r enzyme I (Epstetn etal., 1970).

65

(Continued)


Transport System

Specificity


Gene ptsH

Glucose (via the phosphotransferase system)

Originally characterized as a transport system f o r glucose and alkyl @ - a n dP-glucosides (Englesberg etal., 1961; Hoffee and Englesberg, 1962; Hoffee et a/., 1964; Rogers and Yu, 1962; Hagihara etal., 1963; Winkler, 1971a). N o w k n o w n t o involve a constitutive enzyme II o f the phosphotransferase system.

Fructose (via the phosphotransferase system)

p-Glucosides (via the phosphotransferase system)

umg

ptsF

A l k y l 0 - a n d aryl Pglucosides (Schaefler, 1967; Schaefler and Maas, 1967).

Mannitol, via the phosphotransferase system

bgl6

mtlA

66



Very closely linked t o

Inability t o ferment fructose

ptsl in the orderpurC p tsl ptsH supN ( Ep s t ei n

+ mannose (Fox and Wilson, 19681 or mannitol + sorbitol

eta/., 19701, consistent

(Epstein eta/., 1970).

w i t h the data of Bourd etal. (19681 and Wang etal. (1970). Earlier evidence for close linkage came from mutant strain P34, isolated b y Gershanovitch e t a / . (1967a.b), which maps in the same region and lacks both enzyme I and HPr activities (Bourd e t a / . , 19681; P34 does not revert, so it may be a deletion covering both t h e p t s l and ptsH genes.

Transport Defect in Mutants Defective in the uptake of a variety of sugarsand in HPr (Foxand Wilson, 19681. In a recent study all theptsH mutants isolated were phenotypically leaky,and it was suggested that HPr-which presumably has a very stable tertiary structure, in view of i t s resistance t o heat denaturationmay retain at least partial activity following most amino acid substitutions (Epstein eta/., 1970).

Maps at 23.5 minutes, cotransducible w i t h purB (Kornberg and Smith, 1972).

The growth of E. coli on fructose is normally inhibited b y glucose and, in many strains, by noncatabolizable glucose analogs such as 2sleoxyglucose and amethylglucoside. umg mutants were selected b y the ability t o grow on fructose in the presence of 2deoxyglucose (Kornberg and Reeves, 1972apl.

Defective in the uptake o f glucose and amethylglucoside, and in enzyme II for glucose (Kornberg and Reeves, 1972a,b,cl. Similar mutants have been described b y Schaefler (1967) and Foxand Wilson (19681.

Located between thy and his at about 42 minutes (Ferenci and Kornberg, 19711.

Inability t o grow on fructose as sole carbon source (Ferenci and Kornberg, 1971I.

Defective in fructose uptake and in enzyme II for fructose (Ferenci and Kornberg, 1971I.

Closely linked t o bg/A (the structural gene for aryl P-glucosidesplitting enzyme) and bglC (regulatory gene), between pyrE and ile (Schaefler and Maas, 19671.

Isolated from a P-glucosidefermenting parent strain (bg/B+l b y the inability t o ferment arbutin @-hydroquinonyl~-Dglucoside) (Schaefler, 1967; Fox and Wilson, 19681.

Defective in the uptake o f P-glucosides and in enzyme II for pglucosides (Foxand Wilson, 1968).

71 minutes; cotransducible w i t h lct (Solomon and Lin, 1972).

Inability t o utilize mannitol as carbon source (Solomon and Lin, 1972).

Lack enzyme I I for mannitol (Solomon and Lin, 19721.

(Continued) 67


Transport System

Specificity

fscherichia

Gene

mt/C

coli (Con't.)

Sa/monel/a typhimurium


Very similar to the phosphotransferase system of (see above; Kundig and Roseman, 1 9 7 1 a p ) .

f.coli

carA btsl)

Carbohydrates Method of Isolating Mutants

Linkage


Closely linked t o m t l A (see above) and t o mtlD (thought t o be the structural gene f o r mann i t o l dehydrogenase) (Solomon and Lin, 1972).

Constitutive mutants were isolated b y the ability t o grow on mannitol as sole carbon source,at a concentration near or below the threshold of induction o f the necessary enzymes (Solomon and Lin, 1 9 7 2 ) .

Regulatory gene affecting mtlA (enzyme II f o r mannitol) and mt/D (mann i t o l dehydrogenase) (Solomon and Lin, 1972).

Originally reported t o map nearpro (Levinthal and Sirnoni, 1969); n o w thought t o map near cysA and trzA (Eerkowitz, 1971; Cordaro and Roseman, 1972) The latter position is consistent w i t h the location o f p t s l mutants in E. coli (see above).

Inability t o ferment melibiose (Simoni eta/., 1967; Levinthal and Simoni, 1969; Levinthal, 1971) o r sorbitol (Eerkowitz, 1971 1.

Defective i n the uptake of a variety of sugars and in enzyme I o f the phosphotransferase system (Simoni eta/., 1967; Levinthal and Simoni, 1969; Berkowitz, 1971). As in E. coli, there are t w o classes o f enzyme- I mutants (Saier e r a / . , 1970): (1 ) "Leaky" mutants, containing

0.55% of the wild-type level of enzyme I; these strains grow poorly on glucose, fructose, mannose, rnannitol, sorbitol, and N-acetylglucosamine). (2) "Tight" mutants, containing no detectable enzyme I (less than 0.1% o f the wild-type level); these strains grow poorly on the above sugars and in addition on maltose, lactose, melibiose, and glycerol. They are also reported t o be poorly motile and t o show altered activities o f some membranehound enzymes, suggesting a structural defect in the cell membrane (Saier and Roseman, 19726.b). The sensitivity o f pfs mutants t o carbohydrate repression is discussed b y Saier and Roseman (1972b) and Saier e t a / . (1971).

Adjacent t o carA. in the order cysA rrzA cart? carA (Cordaro and Roseman, 1972).

Inability t o ferment melibiose (Levinthal and Simoni, 1969).

Defective in the uptake o f a variety of sugars and in HPr o f t h e phosphotransferase system (Levinthal and Simoni, 1969; Saier eta/., 1970).

-

(Continued) 69

TABLE I Mutations Affecting Membrane Transport (Continuedl Carbohydrates Organism

Transport System


Mannirol (via the phosphotransferase system)

Aerobacter aerogenes

Specificity

Gene m tl

Deoxyribose

Not characterized.


See description under E. coli.

deo P

Mannitol (via the phosphotransferase system) Fructose (via the phosphotransferase system)

"Enzyme 11" for fructose consists of a high-molecularweight protein (probably constitutive) and a smaller, inducible protein (Km factor), which increases the affinity of the system for fructose, from a K,,, of 20-80 mM t o a K,,, of less than 1 m M (Hanson and Anderson, 1968).

-

Streptococcus lactis strain C2F

Lactose (via the phosphotransfer ase system)

Two wild-type strains of S. lactis appear t o transport lactose b y different mechanisms-strain C2F via the phosphotransferase system, and strain 7962 by some other route (McKayetal., 1970).

-

Streptococcus lactis strain 7962

Lactose

Staphylo. coccus aureus


Similar t o the phosphotransferase system of E. coli, except for the involvement of factor Ill (see below) (Kennedy and Scarborough, 1967; Simoni etal., 1968; Laue and MacDonald, 1968a.b; Hengstenberg et a/., 1968b. 1969).

70

car




Maps at about 115 minutes o n the Salmonella chromosome, close t o the gene for mannitol 1 -phosphate dehydrogenase (Berkowitz, 1971).

Inability t o ferment mannitol (Berkowitz. 1971 ).

Defective in the uptake o f mannitol and in mannitol phosphotransferase (Berkowttz. 1971).

Maps at about 34 minutes (Hoffee, cited in Sanderson, 1970).

Inability t o ferment deoxyribose (Hoffee, 1968).

Defective in the uptake of deoxy ribose (Hoffee, 1968).

Inability t o ferment mannitol (Tanaka and L i n , 1967).

Lack enzyme 1;defecttve in the uptake of D-mannttol (and presumably, on the basis o f growth characteristics, D-sorbitol , D-g lucose , D-mannose, and D-fructose) (Tanaka and L i n , 1967).

Same as above

Lack HPr; same transport defect as enzyme4 mutants (Tanaka and Lin, 1967).

Same as above.

Lack enzyme 11 for D-mannitol (Tanaka and Lin. 1967).

Inability t o ferment fructose (Sapico etal., 1968).

Lack t h e K, factor b u t s t i l l contain t h e l o w a f f i n i t y enzyme II for fructose. Grow slowly on fructose b u t normally on glucose, mannose, mannit o l , and glycerol (Hanson and Anderson, 1968).

Inability t o ferment lactose (McKav e t al., 1970).

Defective in enzyme I1 and factor I l l for lactose; contain normal amounts o f enzyme I and HPr (McKayetal., 1970).

Same as above

Defective in lactose uptake (McKay eta/., 1970).

Inability t o ferment sucrose, mannitol, or maltose (Egan and Morse, 1965a) or sucrose t fructose, or lactose t mannose (Hengstenberg eta/., 1969).

Defective in the uptake o f a variety o f sugars (lactose, sucrose, maltose, galactose, mannitol, mannose, fructose, trehalose, glycerol), all believed to be substrates o f the phosphotransferase system in S. aureus (Egan and Morse,

71

(Continued)


Transport System

Specificity

Gene

Staph ylo coccus aureus ICon't.)

Lactose (via the phosphotransferase system)

S. aureus differs from E. coli in phosphorylating a wider variety o f sugars (including lactose) by means of i t s phosphotransferase system, and also in requiring-in addition t o the sugarspecific, membranebound enzyme Il-a sugar-specific cytoplasmic factor Ill (Simoni er a/., 1968; Hengstenberg eta/., 1969). Factor Illlachas now been purified and characterized (Nakazawa eta/., 1971; Schrecker and Hengstenberg, 1971; Hays and Simoni, 1971).

Pseudo rnonas aeruginosa

Glycerol

Wild-type P. aeruginosa appears to have two inducible systems for the uptake o f glycerol. w i t h Km's of 7.8 x 10-6 M and 4.8 x 1 0 4 M. It is not clear whether uptake occurs by facilitated diffusion (as in E. coli) or b y active transport; an energy requirement has not been demonstrated (Tsay eta/., 1971).

Neurospora crassa

Glucose

Dglucose i s taken up by t w o systems in Neurospora: (1 A high-affinity system. repressed during growth on glucose, with a K,,, of 1-7 x 10-5 M for glucose and 4 x 10-3 M for sorbose. This system is clearly energydependent (inhibited b y azide and dinitrophenol), and can accumulate 3-0-methylglucose as the free sugar against a considerable concentration gradient (KlingmOller. 1967b.c; KlingmOller and Huh, 1972; Scarborough, 1970ap; Schneider and Wiley, 1971a.b.c; Nevilleetal., 1971); (2) A low-affinity system, present constitutively, with a Krn of 8-25 x 10-3M for glucose. Scarborough (1970b) reported very little uptake of sorbose via this system at an external concentration o f 10 mM, but it seems likely that sorbose is a substrate at much higher concentrations (Crocken and Tatum, 1967). The mechanism of uptake by the low-affinity system has not yet been settled. Scarborough (1970a) reported that 3-0methylglucose could not be accumulated against a con-

72

cnc

-

-

sor

~

~

_

_

_



Transport Defect in Mutants 1965a.b; Hengstenberg e f a/., 1968b). and defective i n enzyme I (Hengstenberg e t a/., 1968a. 1969; Simoni e t a/., 1968).

Inability t o ferment lactose

+ mannitol (Hengstenberg eta/., 1969). Like /acY in E. coli, closely linked t o /acZ (the structural gene for pgalactosidase) and lac/ (the regulatory gene).

Inability t o ferment lactose (McClatchy and Rosenblum, 1963; Morseetal., 1968a.b) or lactose + galactose (Hengstenberg eta/., 19691.

Defective in t h e uptake o f lactose and in enzyme Illac( M o m e t a / . , 1968; Hengstenberg eta/., 1968, 1 9 6 9 ) .

Inability t o ferment lactose (Hengstenberg eta/., 1968b, 1969).

Defective in lactose uptake and in factor Illlac(Hengstenberg e t a/., 1968b. 1969).

Method n o t given,

Defective in glycerol uptake (a complete kinetic analysis was n o t reported, so it is n o t clear whether the mutant lacks one of the t w o uptake systems o r whether it lacks b o t h ) . Very l o w levels o f glycerol4inding protein (Tsayetal., 1971).

Resistance t o sorbose, which in hibits the growth o f wild-type Neurospora ( KI ingmll Iler and Kaudewitz , 1966; KI ingmul ler , 1967a).

T w o o f the classes o f sorbose-resistant mutants ( A and B; mapping o n l i n k age groups V I and VII, respectively) were found t o be partially defective in sorbose uptake (Klingmuller, 1967d). Fructosegrown cells (in which the h i g h a f f i n i t y glucosesorbose system should have been derepressed) showed essentially normal K,'s f o r sorbose, b u t a 3 0 4 3 % decrease i n Vmax; glucose uptake appeared normal, however. In view o f the overlapping substrate specificities o f the t w o hexose transport systems in Neurospora, detailed kinetic studies are needed of b o t h repressed (glucosegrown) and derepressed (fructosegrown) mutant cells t o determine which o f t h e t w o systems i s altered and t o what extent.

+ galactose

Six sor genes have been reported, located o n linkage groups I, Ill, V, Vl,and V I I (and one not yet located, b u t not closely linked t o any of the others) (Klingmilller, 1967a).

Defective in HPr (Hengstenberg eta/., 1969). and presumably in the uptake o f the above sugars.

73

(Con tinued)


Transport System

IC0n't.l

Saccharomyces cere visiae

Gene

centration gradient,and concluded that the low-affinity system involved facilitated diffusion. However, Schneider and Wiley (1971a) did observe accumulation of unaltered 3-0-methylglucose against a concentration gradient, inhibited b y azide and dinitrophenol, and concluded that the l o w a f f i n i t y system was capable of active transport.

Neurospora crassa


Specificity

Hexoses

Kinetic evidence indicates that there are at least four constitutive transport systems for hexoses in A. nidulans : (1) glucose (K, = 4-6 x 10-5 M ) , competitively inhibited by glucose analogs (2-deoxy-D-glucose, 3-0-methyl-D-glucose, G-deoxy-D+lucose) and at higher concentrations, by D-galactose (Ki = 1.1 x M ) and D-mannose (Ki = 1.3 x M ) (Brown and Romano, 1969; Markand Romano, 1971). MI, competitively (2) D-galactose (K, = 3 x inhibited by D-fucose (Ki = 2.9 x l o 4 M I , D-glucose (Ki = 5.0 x l o 4 MI, and D-mannose (Ki = 5.9 x l o 4 M (Mark and Romano, 1971). (3) D-fructose ( K , = 2 x l o 4 M ) ,not inhibited by any sugar tested (Mark and Romano, 1971). (4) L-sorbose, competitively inhibited by D-glucose and D-mannose; kinetic constants not reported (Elorza and Arst, 1971).

Lactose

Not characterized

lac-1

Galactose

An inducible system for Dgalactose and its nonmetabolizable analogs D-fucose and L-arabinose. The mechanism of uptake is not clear; various investigators have postulated active transport (de RobichonSzulmajster, 1961 1, phosphorylative transfer (Van Steveninck and Rothstein, 1965; Van Steveninck and Dawson, 1968; Van Steveninck, 1972). and facilitated diffusion (Cirillo. 1968; Kuo e t a/., 1970; Kuo and Cirillo, 1970).

gal2

sorA

i-, C,

~ 1 3 . gal4

74




Maps on linkage group I (Elorza and Arst, 1971).

Resistance to sorbose; must be distinguished f r o m s o r B mutants, in which sorbose resistance results f r o m loss o f phosphoglucomutase (Elorza and Arst, 1971).

Greatly reduced uptake o f Lsorbose, w i t h a slight reduction in the uptake o f D-glucose (Elorza and Arst, 1971). Presumably,sorA mutants are defecfive i n the sorbose transport system described under Specificity, b u t this interpretation is complicated b y the fact that they are also resistant t o 2deoxy-D-glucose (a substrate f o r the glucose transport system; Mark and Romano, 1971).

Maps on linkage group VI (Gajewski etal., 1972).

Inability t o utilize lactose as carbon source (Gajewski e r a / . , 1972).

Greatly reduced uptake o f lactose (Gajewski e t a / . , 19721.

Maps o n linkage group X I I . N o t linked to the structural genes f o r the galactose pathway enzymes, which map in a cluster o n linkage group I I (Douglas and Hawthorne, 1964; Mortimer and Hawthorne, 1966; Bassel and Mortimer. 1 9 7 1 ) .

Can metabolize galactose only at high external concentrations (Douglas and Condie, 1954).

Defective in the uptake o f D-galactose (de RobichonSzulmajster. 1961) and D-fucose and L-arabinose (Cirillo, 1968; Kuo etal., 1970).

Regulatory mutants w i t h altered inducibility o f b o t h t h e galactose transport system and the galactose pathway enzymes: i - a n d C mutants are constitutive, and ga13 and ga14 mutants are noninducible (Douglas and Hawthorne, 1966; Cirillo, 1968).

75

(Continued)


Transport System

Man

Glucose (kidney, intestine)

Specificity Intestine: a Naclependent system for D-glucose, D-galactose, and structurally related hexoses (reviewed by Crane, 1960,1968). K, for D-glucose (in the presence of saturating amounts of Na+) is 1.5 m M in the hamster intestine (Lyon and Crane, 1966) and 4.2 m M in man (Elsasetal., 1970).

Disease Glucosegalactose malabsorption

Kidney: There appear to be two transport systems for glucose in the proximal renal tubule,on the basis of in vivo studies in dogs (Silverman er al., 1970) and man (Elsasetal., 1970). and uptake measurements on isolated brush border preparations from the rabbit (Chesnev, 1971; Busseetal., 19721. System A (Busse eta/., 1972) resembles the intestinal glucose transport system in having a relatively low affinity for glucose (K, = 3 m M in the rabbit) and i n being inhibited b y D-galactose; system B has a higher affinity for glucose (K, = 0.07 mM) and is strongly inhibited b y D-mannose but not b y D-galactose.

Renal glyco. suria

76

Carbohydrates Mode o f Inheritance Autosomal recess1ve (Elsasefal., 1 9 7 0 ) . Intestinal glucose transport is absent in h o m o zygotes and shows a somewhat reduced Vmax ( t o 75% o f the normal value) in heterozygotes. Renal glucose transport is partially impaired in homozygotes and slightly (but probably significantly) lowered in heterozygotes.

Abnormal Phenotype Profuse diarrhea following the ingestion o f glucose, galactose, o r disaccharides containing one or b o t h o f these hexoses. Oral loading with glucose, galactose, or 3-O-methylglucose results in little or n o increase in b l o o d hexose and in the prompt appearance o f large quantities of free hexose in the feces (reviewed in Rosenberg. 1969).

Transport Defect Intestine: Homozygotes appear t o lack the intestinal transport system for glucose and galactose. Biopsy specimens f r o m t w o such patients failed t o accumulate glucose in excess o f the medium concentration (Eggermont and Loeb, 1966; Elms eta/., 1970),and an isolated brush border preparation f r o m another patient did n o t bind Dgalactose-14C o r p h l o r i z i n 3 H (a competitive inhibitor of transport), as shown b y autoradiography (Schneider eta/., 1966). The defect appears t o b e limited t o the glucosegalactose transport system and does n o t extend t o Nadependent transport generally; Eggermont, Meeuwisse, and their colleagues found t h a t 22Na absorption in vivo, and Na,K&imulated ATPase activity and Nadependent amino acid accumulation in vifro, were normal in patients w i t h glucosegalactose malabsorption (Eggerrnont and Loeb. 1966; Meeuwisse and Dahlqvist, 1968). Kidney: The single homozygote studied in detail had a n abnormal renal titration curve f o r glucose, w i t h a reduced minimal threshold. (The maximal reabsorptive capacity could not be determined accurately because the filtered load o f glucose was inadequate). The results are consistent w i t h the view that t h e patient lacked one o f the t w o renal transport systems for glucose (presumably system A ; see Specificity) b u t retained t h e other, h i g h e r a f f i n i t y system (B) (Elsasetal., 1970).

Probably several d i f ferent autosomal recessive mutations. Three pedigrees have been studied in detail (Elsas and Rosenberg, 1963; Elsasefa/., 1971). I n pedigree Holm, the m u -

Appearance o f abnormally large amounts o f glucose in the urine, b u t w i t h no clinical symptoms (reviewed in Rosenberg, 1969)

77

Kidney: T w o types o f renal glycosuria have been distinguished, based o n the shape o f the in vivo glucose titration curves. I n one (type A ) , the curve has a normal shape b u t reaches an abnormally l o w TmG (maximal tubular absorptive capacity f o r glucose); in t h e other ( t y p e 6).glyco-

(Continued]

TABLE I Mutations Affecting Membrane Transport (Continuedl Carbohydrates Organism

Transport System

Specificity

Disease

Man (Con 't.)

Purines and Pyrimidines Organism

Transport System

Specificity

Escherichia coli

Uracil

Not characterized.


Guanine, h y p o xanthine, xanthine

Purine transport in E. coli and presumably in Salmonella is mediated b y membrane-bound phosphoribosyltransferases, and results in the intracellular accumulation o f nucleoside monophosphates. The transport o f guanine and hypoxanthine involves a phosphoribosyltransferase (or possibly more than one) specific f o r the 6-OH purines (Hochstadt-Ozer, 1972a.b). while adenine is transported b y a separate enzyme (Hochstadt-Ozer and Stadtman, 1971a.b.c). Extracellular purine nucleosides are first cleaved to the free bases b y nucleoside phosphorylases and then taken u p b y the same route (Hochstadt-Ozer, 1972a).

78

Gene uraP

-

Carbohydrates Mode of Inheritance

Abnormal Phenotype

tation is clearly i n herited as an autosomal recessive, leading t o mild type-A glycosuria (see Transport defect) in the heterozygotes and severe type-A glycosuria in the homozygote. Pedigrees Cov and Hol can also b e interpreted i n terms of auto. soma1 recessive mutations (one as in pedigree Holm; another producing m i l d type-A glycosuria in the h o m o zygote; and a t h i r d producing type-B glycosuria i n the heterozygote), b u t the data are less complete and the possibility o f autosoma1 dominants w i t h variable expression cannot be ruled o u t

Transport Defect suria begins at an abnormally l o w filtration rate, b u t t h e TmG-when reached-is normal The t w o kinds o f titration curves have been interpreted kinetically t o mean that type A i s the result o f a reduced VmaX of glucose transport, while type B i s the result of an increased K, (Woolf e t 4.. 1966). Intestine: Glucose transport I S normal (Elsas and Rosenberg, 1969). Because renal glycosuria affects the kidney and not the intestine, it is reasonable t o suggest that the defect is in the higher-affinity glucose transport system (system B). In vitro measurements, perhaps w i t h the k i n d o f brush border preparation described b y Busse et a/. (1972). would be required t o test this idea, and t o conf i r m the presence o f a normal system A in patients w i t h renal glycosuria.

Purines and Pyrimidines Linkage Maps at 50 minutes (see Taylor, 1 9 7 0 ) .

-



Resistance t o 6-azauraciI (see Taylor, 1 9 7 0 ) .

Defective in uracil uptake (see Taylor, 1970).

Resistance to 6 m e r c a p t o p u rine or 8azaguanine (Kalle and Gots, 1961).

Defective in uptake o f guanine and hypoxanthine (Zimmerman and Magasanik, 1964). Guanine-hypoxanthine phosphoribosyltransferase activity was present in cell-free extracts f r o m the mutant (Zimmerman and Magasanik. 1964). b u t appeared t o b e an altered enzyme w i t h an abnormal elution pattern f r o m DEAE-cellulose and an abnormal substrate specificity (Adye and Gots, 1966).

Resistance t o 8azaguanine (Thakar and Kalle, 1 9 6 8 ) .

Defective i n the uptake o f guanine and xanthine; purine phosphoribosyltransferases appeared normal (Thakar and Kalle, 1 9 6 8 ) .

(Continued) 79

TABLE I Mutations Affecting Membrane Transport (Continued) Purines and Pyrimidines Organism

Transport System

Specificity

Salmonella typhimurium 1Con't.l

Gene gxu


Purines

Not characterized


Adenine, guanine, hypoxanthine

Not characterized.

Xanthine, uric acid

Not characterized.

uap

ua Y

Saccharornyces cere visiae

Cytosine

Not characterized.

cyt-p, FCY-2

Uracil

There appear t o be t w o uptake systems for uracil in wild-type S. cerevisiae, w i t h Km's o f 5 x 10-6 M and 2.5 x 104 M. (Grenson, 19691.

ura-p, FUR4

80

Purines and Pyrimidines Linkage NearproAB (Benson 8t a/., 1972).

Method of Isolating Mutants 2 3 o f 5 3 p r o A B deletion mutants were found t o be also deleted in gxu, an adjacent gene. The mutants were resistant t o Bazaguanine, and when carrying an additional purE mutation were unable t o use guanine or xanthine as a purine source (Benson 8f a/.. 1972).

Transport Defect in Mutants Defective in uptake and phosphoribosyltransferase activities for guanine and xanthine (Benson 8t a/., 19721. The genetic relationship among these various purine transport mutants remains t o be established.

Resistance t o 8-azaguanine or 6-mercaptopurine (Brockmaneral., 1961).

Lack guanine-hypoxanthine phosphoribosyltransferase; transport not studied (Brockman etal., 1961 1.

Resistance to Bazaxanthine (Brockman etal., 1961 1.

Lack xanthine phosphoribosyltransferase (Brockman eta/., 1961).

Resistance t o Bazsadenine (Brockman etal., 1961 1.

Lack adenine phosphoribosyltransferase (Brockman era/., 19611.

Resistance t o Bazaguanine or t o purine (Darlington and Schazzochio, 1967).

Thought t o be defective in the uptake of adenine,guanine, and hypoxanthine (an indirect argument based on growth data) (Darlington and Schazzochio, 1967).

Resistance t o 2-thiouric acid or Z-thioxanthine (Darlington and Schazzochio, 1967).

Thought t o be defective in the uptake of xanthine and uric acid (an indirect argument based on growth data) (Darlington and Schazzochio, 1967).

Same as above.

Thought t o be a regulatory gene controlling the xanthine-uric acid transport system and also xanthine dehydrogenase and urate oxidase (Darlington and Schazzochio, 1967).

Not yet established whether cyr-p and F C Y Z are allelic.

Inability t o use cytosine as sole source of nitrogen (Grenson, 1969); resistance t o 5-fluorocytosine (Jund and Lacroute. 1970).

Both cyr-p and FCY-2 are defective in cytosine uptake (and have a normal level o f cytosine deaminase, which converts cytosine t o uracil) (Grenson, 1969; Jund and Lacroute, 1970).

ura-p and F U R 4 are allelic (Grenson, 1969).

Resistance t o 5-fluorouracil or, in a cpa strain (which lacks the argininespecific carbarnil phosphate syn-

Defective i n uracil uptake (by both systems) (Grenson, 1969; Jund and Lacroute. 1970).

81

(Continuedl


Transport System

Specificity

Gene

Saccharornyces cerevisiae (Con’t.)

Uridine

N o t characterized.

Adenine, hypoxanthine

Wild-type S. cerevisiae appears t o take u p purines b y t w o systems: an adenine transport system (K, = 1.2 x M I , which also accepts hypoxanthine. pyrimidine (4-APP). and 4aminopyrazolo [3,4-d] some other analogs;and a guanine transport system which accepts hypoxanthine and E-azaguanine (Pickering and Woods, 1972b).

82

wid-p, FUI-1

app-1

Purines and Pyrimidines Linkage



thetase and is sensitive t o inhibition b y uracil, uridine. or cytosine), resistance t o uracil (Grenson, 1969; Jund and Lacroute. 1970). ups and F U R - I are allelic (Grenson, 1969).

Resistance t o uracil o r cytosine (Grenson, 1969); resistance t o 5-fluorouracil (Jund and Lacroute, 1970).

Lack uracil phosphoribosyltransferase activity and show reduced rates o f uracil uptake (Grenson, 1969; Jund and Lacroute, 1 9 7 0 ) . Grenson (1969) has concluded that the effect on uptake is an indirect one-that uracil is accumulated intracellularly b y ups mutants and blocks the transport system b y feedback inhibition. Consistent w i t h this idea, she observed that p y r i midine starvation o f a ups strain resulted in a partial restoration of transport. Hochstadt-Ozer and Stadtman (1971b) have pointed out, however, that t h e amount o f transport was small in these experiments (about 3%o f that seen in other starved strains), that ups strains might have l o w activities of uracil phosphoribosyltransferase (which was not assayed directly), and that an obligatory role o f t h e phosphoribosyltransferase in transport cannot be ruled o u t . In this case the identity of t h e ura-pgene product and i t s role in transport, relative to the phosphoribosyltransferase, w o u l d be interesting t o invest igate.

N o t yet k n o w n whether wid-p and FUI-1 are allelic.

Resistance o f a cpa strain t o uridine (Grenson, 1969); r e sistance t o 5-fluorouridine (Jund and Lacroute, 1970).

Defective in uridine uptake (Grenson, 1969; Jund and Lacroute, 1 9 7 0 ) .

Resistance t o 4-APP (see Specificity; Pickering and Woods, 1972a.b).

Defective in the uptake o f adenine and hypoxanthine. Purine phosphoribosyltransferase activities are higher than normal in app-1 mutants, h o w ever. Either the mechanism of purine uptake is different f r o m that in bacteria (see above) (Pickering and Woods, 1972a.b). or the mutation

-

83

(Continued)


Transport System

Specificity

Gene

Sacchsromyces cerevisiae (Con’t.)

Chinese hamster cells

Thymidine

Not characterized.

TP-

Miscellaneous Compounds Organism

Transport System

Specificity

Escherichia Vitamin 812

Uptake of vitamin 612 by E. coli consists of two

coli

phases: (1) an initial rapid phase which is energyindependent and is thought to represent the binding of 812 to a membrane carrier (Km = 5 x 10-9 M ) ;and (2) a slower, energydependent phase (Di Girolamo and Bradbeer, 1971; Taylor et a/., 1972).

Biotin

Not yet clear whether there is a specific biotin transport system in E. coli, or whether biotin simply diffuses into the cell and is covalently bound (Campbell eta/., 1972).

Thiamine

An energydependent transport system with a Km of 8.3 x l o 7 M, inhibited by the analogs pyrithiamine and oxythiamine (Miyata et a/., 1967; Kawasaki etal., 1969a). Thiamine is normally accumulated as the pyrophosphate, and because the kinases are membranebound, there has been speculation that uptake occurs by group translocation, as in the bacterial phosphoenolpyruvate phosphotransferaseand purine phosphoribosyltransferase systems. Recently, however, mutants lacking thiamine kinase have been shown to take up free thiamine against a concentration gradient, a result that argues against an obligatory role of phosphorylation in transport; likewise, mutants lacking thiamine monophosphokinase can accumulate thiamine mono84

Gene

-

bir

-

Purines and Pyrimidines Linkage


Transport Defect in Mutants has altered the phosphoribosyltransferase, increasing its activity in cellfree extracts while at t h e same time making it unable t o function in transport.

-

Direct autorad lographic screening method (Breslow and Goldsby, 19691.

Defective in thymidine uptake (Breslow and Goldsby, 1969).

Miscellaneous Compounds Linkage

Between argC and thiA, close t o rif (Campbell eta/., 1972).



Isolated, in a parent strain which requires either vitamin 812 or methionine, by t h e inability t o grow at low 612 concentrations in the absence of methionine (Di Girolamo eta/.. 1971).

Defective in the initial rapid binding of 612 (Di Girolamoetal., 1971).

Same as above.

Defective in the slow, energydependent phase of 812 uptake and i n the conversion of 6 1 2 t o other cobalamins (Di Girolamo e t a/., 19711.

Inability of a biotin-requiring strain (bio-) t o grow at low biotin concentrations (Campbell eta/., 1972).

The primary effect of the b i r mutation i s not yet certain. bio-bir- strains are deficient in biotin uptake (or binding); and biot bir- strains are derepressed for at least one biosynthetic Qene (bioD),so that they overproduce and excrete biotin (Campbell eta/., 1972).

Isolated, in a thiamine-requiring strain of E. coli, by the inability t o grow at low thiamine concentrations (Kawasaki eta/., 1969b).

Defective in thiamine uptake ( b u t with elevated amounts of thiamine kinase) (Kawasaki eta/., 1969b).

(Continuedl 85

TABLE I Mutations Affecting Membrane Transport (Continued) Miscellaneous Compounds Organism

Transport System

Escherichia coli (Con’t.)

Specificity phosphate (Kawasaki and Yamada, 1972). A thiaminebinding protein ( K D = 10-7 M ) has been isolated f r o m E. c o l i by osmotic shock; both it and thiamine transport are repressed by growth in the presence o f thiamine (Iwashimaetal., 1971; Kawasaki and Esaki, 1971).

Shikimic acid

Not characterized.

Glycolate

Not characterized.

Bacillus subtilis

Citrate

An inducible transport system for citrate (K, = 2.3 x l o 3 M ) . conveniently studied i n an aconitaseless mutant that cannot metabolize citrate (Willecke and Pardee. 1971a).

Saccharomyces cefevisiae

Ureidosuccinic acid

Ureidosuccinic acid (USA), the first specific intermediate o f the pyrimidine pathway, is transported by a system which i s derepressed in a low-nitrogen medium (e.g., proline) and repressed in a high-nitrogen medium (e.g., ammonium sulfate, glutamate) (Drillien and LaCroute, 1972).


Gene

S-Adenosylmethionine

A transport system for S-adenosylmethionine (K, = 3.3 x l o 6 M ) and S-adenosylhomocysteine, competitively inhibited by S-adenosylethionine (Murphy and Spence, 1972).

Acetate

Not characterized.

shiA

-

weP-1

ure-1, 2.3.4

Sam-pl

-

Miscellaneous Compounds Linkage

Closely linked to his andaroD (Pittard and Wallace, 1966).



Isolated, in an aroD parent strain (unable t o convert dehydroquinate t o dehydroshikimate) by the inability t o grow on shikimic acid. Two mutants were obtained: A82879, which required all the aromatic amino acids and vitamins for growth, and AB2880, which required only shikimic acid and tyrosine (Pittard and Wallace, 1966).

Defective in the uptake of shikimic acid and probably dehydroshikimic acid. There was no measureable uptake in AB 2879, and uptake in A82880 was reduced b y approximately 40% (Pittard and Wallace, 1966).

Ability t o use glyoxylate but not glycolate as sole source of carbon and energy (Ornston and Ornston, 19701.

Defective in the uptake o f glycolate (Ornston and Ornston, 1970).

Two mutants have been isolated with partial defects in citrate transport (McKillen eta/., 1972).

-

Resistance to USA, which inhibits the growth of the wildtype strain o n proline medium (Drillien and Lacroute, 1972).

Defective in USA uptake (Drillien and Lacroute, 19721.

Ability of a pyrimidine-requiring parent strain t o grow on USA in a high-nitrogen mediu m (Lacroute, 1971; Drillien and Lacroute, 1972).

Constitutive for the USA transport system and the general amino acid transport system, both of which are normally repressed in high-nitrogen medium. I t has been postulated that the ure strains are not true regulatory mutants but instead are altered in nitrogen metabolism (Drillien and Lacroute, 1972).

Isolated, in a methioninerequiring parent strain (rnet2a). by the inability t o grow on S-adenosylrnethionine (Spence and Shapiro, 1967).

Defective in the uptake o f S-adenosylmethionine and S-adenosylhomocysteine (Spence, 1971; Murphy and Spence, 1972).

Inability t o grow on acetate (Lanier, 1971).

Defective in the uptake of acetate and probably pyruvate (Lanier, 1971).

87

(Con tin uedl

TABLE I Mutations Affecting Membrane Transport (Continued) Miscellaneous Compounds Organism

Transport System

Man

Vitamin 8 1 2

Spec if ic it y The absorptlon of vitamin 8 1 2 f r o m the intestine requires an ”intrinsic factor” elaborated b y the gastric mucosa.

Disease Heredltary malabsorption o f vitamin B12

Inorganic Cations Organism

Transport System

Escherichia coli

K

Specificity Wild-type E. coli K-12 takes up K b y at least t w o systems:

Gene kdpA, B

C. D

( 1 ) A high-affinity system, which allows cells t o grow rapidly in media containing l o w concentrations o f K (as l o w as 10-5 M ) b u t i s repressed when cells are grown in high-K media (Epstein and Waters, cited in Epstein and Kim, 1971).

(2) A lower-affinity system. thoroughly characterized kinetically. which takes u p K in exchange for Na and H (Schultz e t a / . , 1963). Recently, Bhattacharya e t a / . (19711 showed that membrane vesicles f r o m E. coli B and K-12 can accumulate K (and R b ) in a process that is stimulated by the cyclic depsipeptide antibiotic valinomvcin.

88

(kac)

Miscellaneous Compounds Mode of Inheritance Probably an autosomal recessive (Imerslund, 1960; Spurlinget al., 1964; Mohamed et a/., 1966). In two families parents of affected children have shown normal absorption of vitamin 612 (Gr%beck etal., 1960; Spurling etal., 1964). while in a third family, the parents showed a partial defect (Mohamed eral., 1966).

Abnormal Phenotype Megaloblastic anemia which responds t o parenteral therapy with vitamin 812 but not t o intrinsic factor; persistent proteinuria (not understood) (Imerslund, 1960; Grassbeck eral., 1960; Spurling et al., 1964; Mohamed e t al., 1966). Must be distinguished from classic pernicious anemia in the adult, caused by a deficiency of intrinsic factor.

Transport Defect Presumably a defect in the intestinal transport o f vitamin 612.

Inorganic Cations Linkage Four cistrons, mapping in a cluster in the order DCBA near the gal region, and possibly constituting an operon (Epstein and Davies, 1970).

Method of Isolating Mutants Inability t o grow at very low K concentrations (2 x M) (Epstein and Davies, 1970).

Inability t o grow at low K Three independently concentrations (E. coli 6; isolated kac mutants map near gal (Burmeister, Damadian, 1966, 1968). 1969) and may be allelic with kdp (see above).

trkA and Bare closely linked t o strA; trkC t o pdxA; rrkD t o ilv; and trkE t o trp (Epstein and Kim, 1971). Nocom-

Isolated, from a kdp parent strain (see above), by the inability t o grow at intermediate K concentrations (10-4 t o 5 x 10-3 M ) (Epstein and Kim, 1971).

Transport Defect in Mutants Lack the high-affinity K transport system (Epstein and Waters, cited in Epstein and Kim, 1971).

Defective in K uptake at low concentrations (below lo4 M ) but normal at higher concentrations (Damadian, 1968). May lack the high-affinity K transport system described for E coli K-12 by Epstein and Waters (see above). Also defective in the uptake o f inorganic phosphate (Damadian. 1967). possibly indicating a link between K and PO4 uptake (Weiden e t a l . , 1967). Defective in the lower-affinity system (or systems). trkA, D, and E have reduced rates of K uptake, and t r k 6 and Care defective in the retention o f K (Epstein and Waters, cited i n

(Continued) 09

TABLE I Mutations Affecting Membrane Transport (Continued Inorganic Cations Organism

Transport System

Specificity

Gene


Mg

Kinetic studies of Mg transport in wild-type E. coli cor have not yet led t o a clear picture of the system or systems involved. Km's of 3 x lo4 M t o 5 x lW4 M have been reported for cells grown under various conditions (broth, synthetic medium), and C o and Mn have shown t o inhibit uptake (Silver, 1969;Silver and Clark, 1971; Lusk and Kennedy, 1969;Nelson and Kennedy, 1971). mng Recently, on the basis of studies with Co-resistant mutants (see Transport defect), Nelson and Kennedy (1972)have postulated that wild-type E. coli has two transport systems for Mg:

( 1 ) a constitutive system, competitively inhibited by Co; and (2)a system which is repressed during growth at high Mg concentrations (and has l i t t l e affinity for Co). The kinetic constants of the two systems (K, for Mg. Ki for Co and Mn, VmaXI have not yet been reported.

90

Inorganic Cations Linkage


plementation occurred among 19 trkA mutations or among 6 frkB mutations, indicating that these are probably single cistrons. Not enough trk C, D, and E mutations were available to permit significant comDlementation studies.

Transport Defect in Mutants Epstein and Kim, 1971).Consistent with the whole-cell results, membrane vesicles from a trkA mutant were shown t o have abnormally low rates of valinomycinstimulated K uptake (see Specificity), and vesicles from a trkB mutant were (at least in some experiments) defective in K retention (Bhattacharyaet a/., 1971). t r k D appears t o control a minor transport system (possibly a third, previously undetected system) which is normally overshadowed by the major transport system associated with the rrkA locus; r r k D mutants are isolated only in trkA- strains (Epstein and Kim, 1971 ).

Maps near leu (Lubin Inability t o grow at low K conmay and Kessel, 1960); centrations (€. co/i B; Lubin be allelic with trkC and Kessel, 1960). (Epstein and Kim, 1971).

Defective in the retention of K (Lubin and Ennis, 1964;Lubochinsky eta/., 1964,1966;Gunther and Dorn, 1966;Zimmermann and Pilwat, 1971; Pilwat and Zimmermann, 1972).

Ability to grow in the presence M Co (Nelson and of 5 x Kennedy, 1971,1972).

cor mutants are thought t o lack the constitutive Mg-Co transport system, since they take up very l i t t l e Co and, when grown at high Mg concentrations, very little Mg (Nelson and Kennedy,

1972). Maps between aroD and his (Silver eta/., 1972).

Ability to grow in the presence of M Mn (Silver eta/.,

1972).

mng mutants take up Mg with a normal Km (studied over the range 3 x 10-5t o 1 x lo4 M )but are slightly less sensitive t o Mn inhibition (Silver e t a/., 1972);these experiments were performed with broth-grown cells, which would normally possess both of the Mg transport systems postulated by Nelson and Kennedy

11972). rnng mutants are not resistant t o Co, nor are cor mutants resistant t o Mn (Silver ef a/., 1972).suggesting that they may be defective in different transport systems. This notion could be tested b y a kinetic comparison (over a wide range of Mg, Mn. and Co concentrations) o f the two mutants grown on low and high Mg.

(Continued) 91

TABLE I Mutations Affecting Membrane Transport (Continued) Inorganic Cations Organism

Transport System

Escherichia Fe coli (Con't.)

Specificity E. coli takes up Fe by at least two routes: (1) i n the form of a chelate with enterobactin (also called enterochelin), a cyclic trimer of 2.3-dihydroxyN-benzoylserine (O'Brien and Gibson, 1970). synthesized from chorismic acid via 2.3-dihydroxybenzoate (Young etal., 1971); and (2) as a chelate with citrate (Young et a/., 1967).

Gene entA, 6,

" D'

The former system is induced in Fe-deficient medium (Young et a/., 1967; Brot and Goodwin, 1968; Young and Gibson, 1969b; Bryce and Brot, 1971) and the latter in citrate-containing medium (Cox ef a/., 1970).


Fe

Fe transport i n Salmonella, as in E. coli, involves chelatin with enterobactin (enterochelin) (Pollack and Neilands, 1970) or with a variety of other catechols or hydroxamates (Luckey etal., 1972).

en b class I and I I

sidA-H, J-M

Aerobacter aerogenes

Mg

An energy-dependent transport system for Mg, inhibited by Ni, Co, and (less effectively) Zn (Webb. 1970a).

92

-

~ _ _ _ _

~




entA, B, C, D, €, F, and fep (see below) are closely linked t o one another, and are located between purE and lip (Cox etal., 1970; Youngetal., 1971; Luke and Gibson, 19711, This gene cluster has been postulated to be an operon, repressed in the presence of Fe; the order of genes within the cluster is not known.

Inability t o grow on Fe-deficient medium unles citrate i s present t o induce the alternative system for Fe uptake (Young et al., 1971).

See above.

See above.

fep mutants can synthesize enterobactin, but are defective i n the uptake of the Fe-enterobactin complex (Luke and Gibson, 1971).

enb class I and class I I

enb mutants were originally

enb class-I I mutants d o not accumu-

mutants are closely linked, and located at about 20 minutes on the Salmonella chromosome (Pollack etal., 1970). It is not yet known how many genes are included in the enb region but, as in €. co/i, this is thought to be an operon repressed i n the Dresence of Fe.

isolated by their inability t o grow on minimal medium, by their failure t o respond t o any of the usual growth factors, and by the fact that an unusually large halo of bacteria grew up around each colony of revertants t o wild type (now known t o be due t o the excretion of enterobactin by the revertants) (Pollack etal., 1970).

late any detectable intermediates and are thought t o be blocked in the synthesis of 2.3-dihydroxybenzoic acid; enb class-I mutants accumulate 2.3-dihydroxybenzoic acid and are thought t o be blocked in i t s conversion t o enterobactin (Pollack et al., 1970).

sidA-G and sidM are cotransducible with panC at 9 minutes; sidJ with enb; and sidK and L are unlinked t o either panC or enb (Luckey etal., 1972).

Isolated, in an enb parent strain, b y resistance t o albomycin, a hydroxamate antibiotic (Luckey et al., 1972). The mutants were subdivided into 12 phenotypic classes (sidA-H, J-M) on the basis of their growth responses t o other hydroxamates and catechols.

Appear t o lack various transport systems for hydroxamates and catechols (Luckey etal., 1972).

Ability t o grow in the presence of 10-3 M Co (Webb, 1970b).

Defective in the uptake o f Mg, Co, and Ni (Webb. 1970b).

Defective i n Fe uptake via the enterobactin system. entC mutants are blocked in the first step of enterobactin biosynthesis (the conversion of chorismic acid to isochorismic acid); ent6 mutants in the second step (the conversion of isochorismic acid t o

2.3-dihydro-2.3-dihydroxybenzoic acid); entA mutants in the third step (the oxidation of 2.3-dihydro-2.3dihydroxybenzoic acid t o 2.3-dihydroxybenzoic acid); and entD, €, and F mutants in the last step (the conversion of 2.3-dihydroxybenzoic acid t o enterobactin) (Young e t al., 1971; Luke and Gibson, 1971).

93

(Continued)


Transport System

Hydrogenomonas eutropha

NH4


K, Na, H

Specificity Not characterized.

Wild-type S. faecalis, like many other microorganisms, takes up K in exchange for Na and H (Zarlengo and Schultz, 1966). The overall process appears t o be closely related t o a membrane-bound ATPase: both the ATPase and K transport are inhibited by N,N'dicyclohexylcarbodiimide, Dio 9, and chlorhexidine (Harold e t a/., 1969a.b); both are stimulated during growth on low-K medium (Abrams and Smith, 1971 1; and in DCCD-resistant mutants, both have become insensitive t o DCCD (Abrams e t a/., 1972; Harold and Papineau, 1972b). The ATPase has been purified and extensively characterized by Abrams and his colleagues (Abrams eta/., 1960; 1972; Abrams, 1965; Abrams and Baron, 1967, 1968, 1970; Schnebli and Abrams, 1970;Schnebli e t a/., 1970; Baron and Abrams, 1971). I t has a molecular weight of 385,000, and consists o f six 01 and six (3 subunits, each with a molecular weight of 33,000; and it is attached t o the bacterial membrane by another protein, nectin, of molecular weight 37.000. The precise relationship between the ATPase and cation transport remains unclear. Recently, Harold proposed a mechanism based on the Mitchell model for cation transport in mitochondria (Harold and Papineau, 1972a. b; Harold, 1972):

(1) The primary process is postulated t o be the electrogenic extrusion of H ions from the cell, coupled t o the membrane-bound ATPase and creating a membrane potenti 150-200 m V (inside negative). (2) K is vu.tulated to enter the cell along i t s electrochemical gradient 4n a passive, carrier-mediated process.

(3) Na (moving outward) is exchanged for H (moving inward along i t s electrochemical gradient) in a second passive, carrier-mediated process. The main evidence for this scheme comes from indirect measurements of membrane potential (by means of lipid-soluble cations; Harold and Papineau, 1972a.b) and intracellular pH (by means of the distribution of dimethyloxazolidinedione; Harold e t a/., 1970a); and from the effects of ion-conducting reagents on cation fluxes (Harold and Baarda, 1967a.b; 1968a.b; Harold e t a/., 1970a; Harold and Papineau, 1972a.bl. 94

Gene -

CnK6

TrK8

-

dcc


Mapping techniques are not available for S. faecalis, so the genetic relationship of CnK6 to the other cation transport mutants (see below) has not been established.



Slow growth on NH4 as sole nitrogen source at low p H (Strenkoski and DeCicco, 197 1a).

Defective in the transport o f NH4, and dependent on the diffusion of NH3 (an indirect argument based on growth experiments) (Strenkoski and DeCicco, 1971b).

Ability to survive P32 suicide in low-K medium (see isolation of PO4 transport mutants of S. faecalis) (Harold e t al., 1967). C n ~ mutants 6 require abnormally high K concentrations for growth at pH 6, and might also be isolated in this way.

C n ~ mutants 6 are deficient in the net uptake of K or the lipid-soluble cation DDAt in exchange for H at p H 6 (but, when preloaded with Na, can exchange K for Na at nearly normal rates) (Harold et a/., 1970a; Harold and Papineau, 1972b).

Same as above (Harold and Baarda, 19676). T r ~ mutants 8 require abnormally high K concentrations for growth at p H 8, and might be isolated in this way.

is Uptake of K in T r ~ mutants 8 abnormally sensitive t o inhibition by Na (Harold and Baarda, 1967b).

Inability to grow at low K concentrations (Harold e t a/., 1970a).

Strain 7683 is defective in the uptake of K, tris, or H in exchange for Na (Harold e t a/., 1970a; Harold and Papineau, 1972b).

Resistance t o DCCD (Abrams e t a l . , 1972).

dcc mutants possess an altered membrane ATPase which is not inhibited

b y DCCD. Reconstitution experiments have shown that the purified ATPase and nectin (the protein required t o attach the enzyme t o the membrane) are normal in the mutants, and indicate that the defect must be in a third component (a "carbodiimide-sensitizing factor") of the overall ATPase complex (Abrams et a/., 1972). K uptake and H release b y dcc mutants are resistant t o DCCD. consistent with the postulated role of the ATPase i r i cation transport (Abrams et a/., 1972; Harold and Papineau, 1972b). According t o Harold's hypothesis (see Specificity), C n ~ and 6 dcc mutants are said t o have primary defects in the electrogenic extrusion of H ions;

(Continued) 95


Transport System

Specificity

Gene

Alternatively, one might visualize a direct coupling o f K, Na, and H movements, mediated by a single carrier and driven either by ATP directly (as in mammalian cells) or b y the electrochemical gradient for H ions. As long as the coupling is not obligatory-i.e., as long as the fluxes of K, Na, and H can be dissociated from one another under some experimental conditionssuch a model could account for most of Harold's results with lipid-soluble cations and ion-conducting reagents.

Strepfococcus faecalis (Con't.I

6acillus subtilis

K

Not characterized.

6acillus megaterium

Fe

Wild-type 6. megaterium secretes schizokinen (a secondary hydroxamic acid; structure determined b y Mullis ef a/., 19711, and takes up Fe as the schizokinen-Fe chelate.

-

(SK11)

AAD-1

Sfaphylococcus aureus

Fe

Neurospora crassa

K

Wild-type Neurospora takes up K in exchange for Na and H, in an energy-dependent process (Slayman and Slayman, 1968). A t l o w p H the system obeys standard Michaelis kinetics as a function of the extracellular concentration, with a K,,, of 1.1 x l o 3 M; at high pH, however, the kinetics become sigmoid. The results have been interpreted in terms of two alternative multisite models. (1 The transport system contains a second site which must be filled with a cation (H at low pH, K at high pH) in order for transport t o occur. (21 The transport system i s an allosteric protein consisting o f multiple subunits, each with a binding site for K; cooperative interactions among the subunits give rise to a sigmoid curve at high pH, but H serves as an allosteric activator of the system, causing a shift toward a standard Michaelis curve at low p H (Slayman and Slayman, 1970).

96

frk-1



Transport Defect in Mutants strain 7683 in the passive exchange of H for Na; and T r ~ in 8 the passive uptake of K. According t o the singlepump hypothesis, strains C n ~ and 6 7683 are primarily defective in efflux sites, and T r ~ in 8 influx sites.

Inability t o grow at low K concentrations (Lubin, 1964).

Oefective in the retention o f K (Willis and Ennis, 1968).

Requirement for schizokinen for growth (Arceneaux and Lankford, 1966).

Blocked in the biosynthesis of schizokinen and in the uptake of Fe b y this route (Arceneaux and Lankford, 1966; Davis etal., 1971; Davis and Eyers, 1971).

Isolated, in strain SKI1 (see above), b y resistance t o the ferric hydroxamate antibiotic A22765 (Davis and Byers, 1971).

Defective in the uptake of some Fe chelates (A22765, Desferal) but n o t in the uptake of others (schizokinen) (Davis and Eyers, 1971).

-

Resistance t o the ferric hydroxamate antibiotic A22765 (Kniisel etal., 1969).

Defective i n the uptake of Fe chelates such as A22765 (Zimmermann and Kniisel, 1969).

Linkage group I l l (Slayman and Tatum, 1965).

Inability t o grow at low K concentrations; selected by inositolless death or filtration enrichment (Slayman and Tatum, 1965; Slayman and Kopsack, unpublished results).

Oefective in K influx (Slayman and Tatum, 1965; Slayman, 1970, and unpublished results). The best-studied trk-1 mutant, strain R2449, appears t o have a qualitatively altered transport system, with an abnormally low affinity for K and also an abnormal p H dependence of transport. According t o both the allosteric and the two-site models (see Specificity), the higher K requirement o f the mutant would reflect an increase in the K,,, of the transport site(s). The abnormal p H dependence would come from an altered equilibrium between the t w o conformations of the carrier, in the allosteric model, or from altered affinities of the modifier site for H and K, in the two-site model (Slayman, 1970).

97

(Continued)


Transport Svstem

Specificity

Neuro-

Gene

trk-2

spora crassa (Con 't.)

Organism

Transport System

Sheep

Na, K

Specificity Sheep red b l o o d cells, like most other animal cells, transport K inward and Na outward in an ATPdependent process catalyzed b y an (Na + K)-activated membrane ATPase. The system is specifically inhibited b y cardiac glycosides such as ouabain (reviewed b y Glynn, 1968; Skou, 1971).

98

Gene

L/ h (fTIL/M)

Inorganic Cations Linkage Linkage group V (Slayman and Kopsack, unpublished results).

Mode o f Inheritance The L / h gene controls the K concentration o f sheep red cells, w i t h L (low-po tassi u m ) almost completely dominant over h (high-potassium) (Evans and King, 1955; Evans ef a/., 1956; Kidwell ef a/., 1958; Rasmusen and Hall, 1966; Tucker and Ellory, 1970; Eagleton ef a/., 1970). I n addition, a correlation exists between potassium types and the M f L blood group system such that H K sheep (hh) are homozygous for the gene controlling the M antigen (MM), homozygous L K sheep ( L L ) are homozygous f o r the gene controlling the L antigen (mLmL1, and heterozygous L K sheep are M m L (Rasmusen and Hall, 1966, 1967;Tucker, 1968; Lauf and Tosteson. 1969; Ellory and Tucker, 1969a; Rasmusen, 1969). I t is n o t yet clear that L / h and m L l M are identical, and i n fact several lines o f evidence suggest that they may be closely linked b u t separate genes: (1) In reticulocytes f r o m genetically L K sheep, mL is expressed (that is, L antigen is present) b u t L is n o t (the cells contain a high potassium

Method o f Isolating Mutants Same as above.

Transport Defect in Mutants Defective in t h e retention o f K. w i t h an abnormally rapid K - K exchange (Slayman, unpublished results).

Abnormal Phenotype Red cells f r o m H K sheep contain about 80 mmoles K/liter and those f r o m L K sheep, about 1 3 mmolesfliter. The Na concentrations are complementary t o the K concentrations, so that the sum o f Na t K is the same in the t w o cell types; CI concentrations, water content, and cell volumes are also the same, as are plasma K and Na concentrations (Tosteson and Hoffman, 1960). The difference between H K and L K is conspicuous o n l y in mature red cells f r o m adult animals. In genetically L K lambs, and in adult L K animals that have undergone massive hemorrhage so that large quantities o f new cells are entering t h e circulation, the red cells contain high K concentrations (Lee eral., 1966; reviewed in Tucker, 1971).

99

Transport Defect Mature H K and L K cells differ in two main respects w i t h regard t o cation movements across their membranes: ( 1 ) B o t h cation transport and the (Na t K)-stimulated ATPase are less active in L K cells-4 t o 8-fold (Tosteson and Hoffman, 1960; Tosteson, 1963; Dunham and Hoffman, 1971a; Hoffman and Tosteson, 1971) and 4- t o 13-fold (Tosteson e t a / . , 1960; Tosteson, 1963; Whittington and Blostein, 1971 ), respectively. (2) Conversely, the passive permeability t o K ( K leak) is 2 t o 5 times greater in L K cells (Tosteson and Hoffman, 1960; Dunham and Hoffman, 1971a). Perhaps the most interesting feature o f this system is that treatment o f L K cells w i t h anti-L serum (formed in an H K sheep against L K red cells) causes a dramatic stimulation o f cation transport, w i t h a 2 t o 5-fold increase in active potassium influx (Ellory and Tucker, 1969a. 1970a; Lauf e f a/., 1970; Ellory eral., 1972) and in (Na + K)-stimulated ATPase activity (Ellory and Tucker, 1969a). Part o f the difference between H K and L K cells, and also part o f t h e stimulating effect o f anti-L, can be accounted f o r b y a conversion o f p u m p sites t o leaks during the maturation o f the L K cell, and a reconversion t o pumps in the presence o f anti-L. Using ouabain-3 binding as an assay, Dunham and Hoffman (1971a.b) f o u n d 4 2 p u m p sites per H K cell and 7.6 per L K cell la ratio o f about 6 : l . consistent w i t h the ratios o f cation transport and ATPase activity), and an intermediate

(Continued)


Transport System

Specificity

Sheep (Con‘t.I

100

Gene

Inorganic Cations Mode of Inheritance

Abnormal Phenotype

concentration) (Ellory and Tucker, 1 9 7 0 ~ ) . (2) I n adult L K cells, where both are expressed, it is possible to distinguish two responses t o anti-L antiserum-a marked stimulation of K transport (see Transport defect) as well as complement-mediated immune hemolysis. The first response is abolished i f the cells are pretreated with trypsin but the second i s not, a result most easily interpreted i n terms of separate L and m L gene products (Lauf etal., 1971). (3)Finally, goat red cells also have the H K I L K transport system and M I L blood groups, but in this case the two are clearly controlled by unlinked genes; sheep anti-L serum stimulates transport in L K goat cells, pointing to a close relationship between the L genes of the two species, but does not hemolyze goat cells, suggesting that the mL genes may be different (Ellory and Tucker, 1970b).

Transport Defect number of pump sites in cells from L K lambs. I n addition, when L K cells were treated with anti-L, there was a 2-fold increase in ouabain-binding sites (Lauf et a/,, 1970; Ellory etal., 1972) and, at least in one case, a concomitant decrease in the K leak (Ellory etal., 1972; but see Lauf etal., 1970). These quantitative changes can be interpreted in terms of t w o general hypotheses concerning the nature o f HK and L K transport systems. On the one hand, one might imagine that the L gene controls a labile element required for transport, and that the element loses activity during maturation of rhe L K cell but can be reactivated by antiserum. (In what may be a parallel case, several mutant forms of the enzyme p-galactosidase in E. coli are known to be reactivated by antibody; Rotman and Celada, 1968; Celada et sl., 1970; Messer and Melchers, 1970.) The dominance o f L over h, according to this hypothesis, could result from preferential incorporation of the defective L element into the membrane, or from interactions among defective and normal subunits in an oligomeric protein (see discussion of dominance and recessiveness in the text). Alternatively, the L K phenotype might reflect the delayed synthesis of an inhibitor of transport, with antibody somehow masking or inactivating t h e inhibitor. In this case the dominance of L could b e explained if h were a mutant allele, coding for a defective inhibitor. Either hypothesis would have t o account for the recent demonstration that there are qualitative as well as quantitative differences between HK, LK, and anti-L treated L K cells: in their relative affinities for K and Na (Hoffman and Tosteson, 1971; Ellory e t a/., 1972) and ouabain (Dunham and Hoffman, 1971a1, and in some of the partial reactions of the ATPase (Whittington and Blostein,

(Continued) 101

TABLE I Mutations Affecting Membrane Transport (Continuedl Inorganic Cations Organism

Transport System

Specificity

Disease

Sheep (Con 't.)

Man

Hereditary spherocytosis

Na (passive per meabili ty 1

Deer mouse IPeromyscus maniculatus)

Man

Hereditary spherocytosis

Na. K

ATPasedeficient hemolytic anemia

H (passive permeabi Iity )

Renal tubular acidosis

102

Inorganic Cations Mode of Inheritance

Abnormal Phenotype

Transport Defect 1971; Blostein eta/., 1971; Blostein and Whittington, 1972). It would be interesting to know whether these kinetic differences are present at the reticulocyte stage or whether they appear-along w i t h the change in number of pump sites-as the red cell matures.

Autosomal dominant (reviewed b y Jandl and Cooper, 1972).

Hemolytic anemia with jaundice and splenomegaly. The circulating red cells tend to assume a spherical shape, and are more readily sequestered in in the spleen and hemolyzed. The red cells also show an abnormal osmotic fragility, and undergo premature autohemolysis when incubated in vitro (reviewed b y Jandl and Cooper, 1972). A similar but less severe condition involving elliptical red cells has been described b y Honig e t a/., (1971).

Increased permeability t o Na, leading to a compensatory increase in (Na + KI-activated ATPase and-in the absence of glucose-to swelling and hemolysis (Jacob and Jandl, 1964). Believed t o be a primary defect in t h e microfilaments of the red cell membrane. Proteins extracted from the membranes of hereditary spherocytes are abnormal in solubility and in aggregation as a function of ionic strength (Jacob e t a / . , 1971I. In addition, brief exposure of normal red cells t o vinblastine, colchicine, or strychnine-compounds known t o precipitate microfilament proteinsgenerates cells very similar t o hereditary spherocytes in morphology, osmotic fragility, and permeability t o Na (Jacob e t a / . , 1972).

Autosomal recessive (Huestis and Motulsky, 1956).

Hemolytic anemia with jaundice and splenomegaly. Spherical red cells (Anderson e t a/., 1960).

Believed t o be analogous t o hereditary spherocytosis of man.

Autosomal dominant with variable expression (data from two families; Harvald e t a/., 1964).

Hemolytic anemia, with red cells of normal morphology (Harvald e t a / . , 1964).

Postulated t o be a defect in the (Na + K)-activated ATPase and in the transport of Na and K. The data are incomplete, however, since only total red cell ATPase [(Na + KI-activated and Mg-ATPase] was assayed, and since cation fluxes were not studied (Harvald eta/., 1964).

Can be inherited as an autosomal dominant, with variable expression in some families and full expression in others. Most cases of renal tubu-

Sustained metabolic acidosis, with a low concentration of bicarbonate and an elevated concentration of chloride in the plasma. In many patients there are additional distur-

Thought t o be a primary defect in the ability t o generate steep H ion gradients between blood and urine, possibly because the distal tubular cells are abnormally permeable t o H ions. Bicarbonate reabsorption and ammonia

(Continued) 103


Transport System

Specificity

Disease

Man (Con 't.I

Inorganic Anions Organism ~~~

Transport System

Specificity

Gene

~

Escherichia coli

PO4

There are a t least two transport systems for phosphate in E. coli (Bennett and Malamy, 1970a,b, 1971; Medveczky and Rosenberg, 19711: (1) A high-affinity system with a Km of 7 x 10-7 M, inhibited by arsenate. This system appears to involve a phosphatebinding protein,Kg = 8 x 10-7 M . which has a molecular weight of 42,000 and binds one phosphate per molecule of protein (Medwczky and Rosenberg, 1989, 1970). Uptake requires K (Weiden e t a / . , 1967; Medveczky and Rosenberg. 19711 and is regulated by the intracellular pool of inorganic PO4:

(2) A lower-affinity system with a K, of 9.2 x 10-6 M, inhibited by Ni2+ but not by AsO4. Bennett and Malamy (1971) have discussed the conditions under which the two systems are formed, and have reported that during growth in the presence of gluwsed-phosphate, an additional POq-As04 transport system is induced.

104

pitA

Inorganic Cations Mode of Inheritance ~~

Abnormal Phenotype ~~

Transport Defect

~

lar acidosis have negative family histories, however, and the syndrome may arise secondary to pyelonephritis, acquired hyperglobulinemia, or drug therapy (reviewed b y Seldin and Wilson, 1972).

bances of electrolyte metabolism, including an excessive urinary loss of potassium (leading t o hypokalemia, with weakness or paraiyiis) and calcium and phosphate (leading to osteomalacia, nephrocalcinosis, and renal stones) (reviewed by Seldin and Wilson, 1972).

excretion are normal (reviewed by Seldin and Wilson, 1972).

Inorganic Anions Linkage Maps between x y l and malA (Bennett and Malamy, 1970a).

Method of Isolating Mutants Resistance t o As04 (Bennett and Malamy, 1970a.b); slow growth at low PO4 concentrations (Medveczky and Rosenberg, 1970).

Transport Defect in Mutants Defective in the high-affinity phos. phatearsenate transport system. The original pitA mutant (UR13). isolated and mapped b y Bennett and Malamy (1970a,b), was unable t o accumulate A S 0 4 and took UP PO4 at a reduced rate. Mutants 10-1 and 20-2 (Medveczky and Rosenberg, 1970,1971) have been characterized in greater detail; they lack both the high-affinity phosphate transport system and the phosphate-binding protein. and retain only the low-affinity system. Further experiments are needed t o see whether mutants 10-1 and 20-2 map at the pitA locus.

Resistance to A s 0 4 in the presence of La-glycerophosphate as phosphate source (Bennett and Malamy, 1970a,b); resistance to AS04 (Medveczky and Rosenberg, 1970. 1971).

Possible defects in the low-affinity phosphate transport system: Strain U R I (Bennett and Malamy, 1970a.b) appears t o contain at least two mutations-in pitA and in one or more unlinked genes-and transports phosphate at about 10% of the normal rate; it may be defective in both phosphate transport systems. Strain 20-1 (Medveczky and Rosenberg, 1971) lacks the low-affinity

(Continued) 105

TABLE 1 Mutations Affecting Membrane Transport (Continuedl Inorganic Anions ~

~~

Organism

Transport System

Specificity

Gene

Escherichia coli (Con?.]

so4

N o t characterized,

cysP

cysB

cys€


SO4

A transport system f o r inorganic sulfate (K, = 3.6 x cysA 10-5 M I , inhibited b y sulfite, thiosulfate, and chromate. Repressed during growth on cysteine; derepressed during growth on Ldjenkolate (Dreyfuss, 19641. A sulfatebinding protein which may be involved in transport has been crystallized and extensively characterized (Pardee, 1967; Langridgeet a/., 1970).The protein is located in the periplasmic space (between the cell wall and the cell membrane) (Pardee and Watanabe, 1968) and i s released during protoplast formation (Dreyfuss and Pardee, 1965) or osmotic shock (Pardee, 1966). I t has a molecular weight o f 32,000 and binds sulfate w i t h a dissociation constant on the order o f 10-7 M, depending on the ionicstrength (Pardee, 1966). Binding is inhibited b y sulfite, thiosulfate, and chromate, and synthesis o f the binding protein is repressed during growth on cysteine (Pardee er a/., 1966).

106

Inorganic Anions Linkage


Transport Defect in Mutants phosphate transport system and i s derepressed for the high-affinity system; it has not been mapped. Defective in SO4 transport and sulfite reductase (Jones-Mortimer, 1968).

About 5 3 minutes (Jones-Mortimer, 1968; Taylor, 19701

Require cysteine for growth (Jones-Mortimer , 1968).

25 minutes (Yanofsky and Lennox, 1959; Signer et a/., 1965; Taylor, 1970).

Same as above.

72 minutes (JonesMortimer. 1968; Taylor, 1970).

Same as above

A second group of pleiotropic mutations affecting SO4 transport and cysteine biosynthesis (JonesMortimer, 1968).

76 minutes (Mizobuchi e t a/., 1962; Demerec etal., 1963; Sanderson,

Require cysteine for growth; must be distinguished from other cys- mutants that are defective in cysteine biosynthesis (Mizobuchi eta/., 1962; Dreyfussand Monty, 1963).

Defective in sulfate uptake (Dreyfuss, 1964; Ohtaetal., 19711.cysA is not the structural gene for the sulfatebinding protein, however: (1) Although many chromate-resistant cysA a, b, or c mutants produce abnormally low amounts of binding protein, the protein appears to be identical to that of the parent strain as tested by acrylamide gel electrophoresis, immunodiffusion, and heat stability. (2) Two mutants with deletions covering the entire cysA region produce the binding protein in normal amounts. (3)Nonsense mutants in each of the three cysA cistrons produce the binding protein in normal amounts. Ohta et a/. (19711 concluded, on the basis of this evidence, that cysA determines some other component of the transport system, and in addition exerts a regulatory effect (seen in some cysA mutants but not others) on the production of binding protein. The binding protein itself may be necessary for transport, but seems to saturate the system in low amounts (i.e., well before it is produced in maximal quantities, as in derepressed cells). The structural gene for the binding protein has not been identified,

1970).

Pleiotropic mutations affecting

SO4 transport and cysteine biosynthesis (Jones-Mort imer, 1968).

Resistance to chromate (Pardee 8t a/., 1966; Ohta etal., 1971). Most chromateresistant mutants are cysA, but some are in other cys regions. Ability to use Lcysteine sulfinic acid but inability to use either sulfate or thiosutfate as sulfur source; 52 strains isolated in this way were all cysA mutants (Ohta etal., 1971).

107

(Con timed)

TABLE 1 Mutations Affecting Membrane Transport (Continued) Inorganic Anions Organism

Transport System

Specificity

Gene cysB


Salmonella pullorum

SO4

Not characterized.

Pseudomonas pseudomallei

As03 (PO41

Not characterized


PO4

Wild-type S. faecalis has two transport systems for inorganic PO4: one (the 01 system) with a pH optimum of 8-9 and a K, of approximately 10-5 M PO4 or AsO4; and the other (the p system) with a pH optimum of 5.5 and a K, that rises rapidly with increasing pH (Harold etal., 1965; Harold and Eaarda, 1966).

-

Bacillus cereus

PO4

A system that transports phosphate (K, = 3.5 x 10-5 M ) ,pyrophosphate, phosphite (Km = 4.8 x l o 4 M ) , and arsenate (K, = 2.4 x 10-5 M ) (Rosenberg and LaNauze, 1968; Rosenberg et al., 1969).

-

Neurospora crassa

SO4

Wild-type Neurospora has two transport systems for inorganic sulfate (Marzluf, 1970a.b; Roberts and Marzluf, 1971): System I, which predominates in conidia, has Km's of 2 x 104 M for sulfate and 4 x 1 0 6 M f o r chromate; and

108

cys-13

cys-14

Inorganic Anions Linkage 5 2 minutes (Mizobuchi eta/., 1962; Demerec e t a / . , 1963; Sanderson, 1970).



Require cysteine for growth (Mizobuchi et a/., 1962). Resistant t o chromate (Ohta eta/., 1 9 7 1 ) .

A regulatory gene; mutants have reduced levels o f the enzymes o f the cysteine pathway, including the binding protein. A temperaturesensitive c y s 6 mutant was shown t o produce binding protein o n l y a t the lower temperature, b u t once p r o duced, the protein had normal heat stability (Ohtaetal., 1971).

Strain MS35 was one o f 45 isolates o f S. pullorurn that required cysteine for growth (Kline and Schoenhard, 1970).

Strain MS35 was f o u n d t o contain t w o mutations, one leading t o a defect in SO4 transport and the other leading t o temperature sensitivity o f sulfite reduction (Kline and Schoenhard, 1970).

Resistance t o arsenite (Arima and Beppu, 1964).

Decrexed permeability t o arsenite (an indirect argument) (Arima and Beppu, 1964).

32P suicide. After treatment w i t h a mutagen, cells were grown briefly in medium containing 32PO4 of high specific activity,and then stored at -BOO for 30 days; at that time the majority o f cells had been inactivated b y decay o f the 32P incorporated into nucleic acids, b u t transport mutantsunable t o grow on l o w concentrations o f phosphatesurvived selectively (Harold e t a / . , 1965).

Defective in PO4 and A s 0 4 uptake at high pH; thought t o lack t h e a system (see Specificity) (Harold era/., 1965; Harold and Baarda, 1966).

Resistance t o PO3 in the presence o f aminoethylphosphonate as a source of P (Rosenberg and LaNauze, 1968).

Defective in the uptake o f phosphate, phosphite, and arsenate (Rosenberg and LaNauze, 1968).

Maps o n linkage group I, t w o units t o the right o f his-3 (Marzluf, 1970a).

Resistance t o chromate i n the presence o f methionine as S source (Marzluf, 1970a).

Lack transport system I for sulfate and chromate (Marzluf, 1970a.b; Robertsand Marzluf, 1971).

Maps on linkage group IV, approximately 21 units f r o m cot-1 (Marzluf, 1970al.

Originally isolated as chromate-cesistant, b u t when retested, f o u n d t o be sensitive (Marzluf, 1970a).

Lack transport system II for sulfate (Marzluf, 1970a.b).

109

(Continued)

TABLE 1 Mutations Affecting Membrane Transport (Continued) Inorganic Anions Organism

Transport System

Specificity

Gene

System II, which predominates in growing hyphae, has a K,,, of 8 x 106 M for sulfate.

Neurospora crassa (Con 't.I

Both transport systems, along with a group of related enzymes (aryl sulfatase, choline sulfatase, and cholineOsulfate permease1,are repressed during growth on methionine (Metzenberg and Parson, 1966);and both systems, together with the same group of enzymes, are under the control of the cys-3 locus (Marzluf and Metzenberg, 1968)and thescon locus (Burton and Metzenberg. 1972).

cys-3

scon

There appear to be two distinct transport systems for PO4 in Neurospora (Lowendorf & Slayman, 1970 and manuscripts in preparation; Lowendorf,

nuc-I

19721: (1) A low-affinity system is present in cells grown on the standard minimal medium (Vogel's; 37 mM PO4) and possesses rather complex kinetics. The K,,, of this system increases from less than 2 x 10-5M a t pH 4 to 9 x 104 M a t pH 7.2,while the Vmax remains nearly constant at 1.5-1.6mmoles/liter cell water per minute. The results can be accounted for by assuming that H (or OH) serves as a modifier of transport.

nuc-2; pcon

(2)In additi0n.a high-affinity system is derepressed in cells that have been grown on limiting phosphate.This system has simpler kinetics, with a K,,, of about 2 x 10-6M and a VmaXof 4 to 5 mmoles/liter cell water per minute over the entire pH range tested. I t s formation is blocked by cycloheximide.


SO4

A transport system for SO4 (K,,, = 7.5 x los MI, S203(Km=7.5x105MI.Se04(Krn=7.7x105M).

s -3

and MoO4, repressed during growth on methionine (Tweedie and Segel, 1970;Bradfield eta/., 1970).

A. C

110

Inorganic A n io n s Linkage

M e t h o d o f Isolating Mutants


Maps o n t h e left a r m of linkage group II (Metzenberg and Ahlgren, 1970).

Require cysteine f o r g r o wt h .

A regulatory gene w h i ch exerts positive control over the synthesis o f b o t h sulfate permeases, a r yl sulfatase, choline sulfatase, and c h o l i n e a sulfate permease; cys-3- strains d o n o t make any o f these enzymes (Marzluf and Metzenberg, 1968; Metzenberg and Ahlgren, 1971) ,

Maps on t h e right a r m of linkage group V, b e yo nd his-6 (B urton and Metzenberg, 1972)

Hydrolysis o f in d o x y l sulfate (producing a blue color) during g r o wt h in the presence o f m e thionine and sulfate ( B u r t o n and Metzenberg, 1972).

A second regulatory gene f o r th e proteins controlled b y cys-3 (see above); scone mutants are derepressed f o r all these proteins ( Bu r to n and Metzenberg, 1972).

Linkage group I (Ishikawaetal., 1969).

I n a b ilit y o f wild-type Neurospora t o grow o n R N A , o r i n a b ilit y of an adenine-requiring strain t o use R N A as a source o f adenine (Ishikawa eta/.,

Postulated t o be a regulatory gene fo r phosphorus metabolism, analogous t o cys-3 in th e sulfur system (see above) (Lehman e t a/.. 1973). nuc-l mutants cannot be derepressed f o r PO4 transp o r t at high p H o r fo r acid and alkaline phosphatase (Lehman eta/., 1973; T o h e and Ishikawa, 1971 ) .

1969).

Linkage group I I (Ishikawa era/., 1969).

nuc-2/pcon is postulated t o b e a

Same as above

second regulatory gene f o r phosphorus metabolism, analogous t o scon (see above) (Lehman etal., 1973).pconc mutants are derepressed fo r PO4 at high p H and f o r alkaline phosphatase (although not f o r acid phosphatase), while nuc-2 mutants are repressed f o r these activities (Lehman eta/., 1973; Toh-e and Ishikawa, 1971). See, however, Hasunuma and lshikawa (1972) fo r a discussion o f an alternative hypothesis-that nuc-1 and nuc-2 are structural genes f o r a nuclease.

Maps o n linkage group V I (Dorn, 1967).

I n a b ilit y t o g r o w on SO4 as sole source o f sulfur ( Do r n ,

1967). I n a b ilit y t o g r o w on SO4 as sole source of sulfur (Hussey etal., 1965).

Defective in SO4 transport (indirect argument based o n g r o w th experiments) ( Ar st. 19681. Defective in SO4 transport (Spencer eta/., 1968).

(Continued) 111

TABLE 1 Mutations Affecting Membrane Transport

(Continued)

Inorganic Anions Organism Aspergillus nidulans (Con%) Penicillium notaturn

Transport System

Specificity

Gene Gamma

SO4

Organism

Transport System

Man

PO4

Similar t o that of A. nidulans (see above; Tweedie and Segel, 1970;Bradfield etal., 1970).

Specificity Kidney: Evidence has been presented f o r t w o PO4 transport systems in the human kidney (Glorieux and Scriver, 1972):one accounting f o r about t w o thirds o f the total net reabsorptive capacity, regulated b y parathyroid hormone (PTH),and the other,accounting f o r the remaining reabsorptive capacity, regulated b y Ca.

38632M

Gene Familial h y pophosphatemic rickets

Intestine: Likewise, there appear t o be t w o PO4 transport systems in the jejunal mucosa, w i t h apparent Km's o f 6 x 1 0 - 6 a n d 6 x lo4 M (Short etal., 1973).

CI

N o t characterized

112

Congenital chloridorrhea

Inorganic Anions Linkage


-

-

-

-

Transport Defect in Mutants Defective in SO4 transport (Bradfield eta/., 1970). Defective i n t h e transport o f SO4,

S2O3,SeO4, and M o o 4 (Tweedie and Segel, 1970).

Linkage

Abnormal Phenotype

X-Linked dominant (Wintersetal., 1957, 1958; Graham et a/., 1959; Burnett eta/., 1964).

L o w concentration o f i n organic phosphate in the serum, associated w i t h increased urinary excretion o f phosphate; in some cases reduced gastrointestinal absorption of Ca and high incidence o f rickets, not responsive t o physiological amounts of vitamin D (reviewed b y Rosenberg, 1969; Williams and Winters, 1972).

Transport Defect Defective in the PTH-regulated PO4 transport system in the kidney (Glorieux and Scriver, 1972) and in the h i g h a f f i n i t y PO4 transport syst e m in the jejunal mucosa (Short e t a/., 1972).The latter defect, b y leading t o the formation o f insoluble calcium phosphate complexes i n t h e intestinal lumen, could account f o r the reduced absorption of Ca in t h e intestine (Rosenberg, 1969).

A n alternative theory o f familial hypophosphatemic rickets-that the primary lesion is i n the conversion o f vitamin D t o i t s active metabolite(s) (DeLucaetab, 1967; Avioli era/., 19671, leading indirectly t o decreased intestinal Ca transport, secondary hyperparathyroidism, renal loss of PO4, hywphosphatemia, and bone disease-now seems unlikely t o be correct. Hypophosphatemia frequently occurs w i t h o u t any detectable impairment o f Ca absorption, and in addition, it has recently been demonstrated that in patients w i t h untreated hypophosphatemia, serum levels of P T H are normal (Arnaud eta/., 1971). Probably autosomal recessive (Perheentupa eta/., 1 9 6 5 ) .

Diarrhea; high fecal CI concentrations (and almost complete absence of CI in the urine); metabolic alkalosis (Darrow, 1945; Gamble eta/., 1945).

113

Thought t o be a defect in t h e absorpt i o n o f CI i n t h e colon and terminal ileum (Evanson and Stanbury, 1965). Direct f l u x measurements have n o t been performed.

(Continued)

TABLE 1 Mutations Affecting Membrane Transport (Continued) Multiple Transport Systems or General Membrane Permeability Organism Escherichia

coli

Membrane Property

Gene

Several mutants o f €. coli have been described w i t h multiple transport defects. In some cases the energy supply t o a large number o f transport systems has been blocked [e.g., mutants lacking Ca,Mg-ATPase (Eutlin etal., 1971 ;Simoni and Shallenberger, 1972; Schairer and Haddock, 1972) o r mutants lacking enzyme I o r HPr of the phosphoenolpyruvate phosphotransferase system] . Others may be cases i n which the transport systems cannot be formed or are.not fully functional, because of an alteration in membrane structure [the kmt strains o f von Meyenburg (1971) or the mutants of Crandall and Koch ( 1 9 7 1 ) l .

kmt

In addition, a large number o f mutants are k n o w n w i t h abnormal permeability, usually t o a variety o f chemically unrelated compounds. These are almost certainly cellsurface mutants, b u t because o f the complexity o f the surface layers o f €. coli [which include an outer membrane w i t h attached lipopolysaccharides, a rigid peptidoglycan layer, and a plasma membrane (see, for exarnple,Schnaitman, 1971a,b)l, it is n o t always possible t o say which portion o f the surface has been altered.

acrA

envA

114

Multiple Transport Systems or General Membrane Permeability Linkage Between s t r A and metB (von Meyenburg, 1971).


Transport Defect

Delayed growth at l o w glucose Appear t o transport a variety o f concentrations (von Meyenburg, amino acids, carbohydrates, PO4, and SO4 w i t h 20-500-fold increased Km's 1971). (measured indirectly). Postulated t o have an altered cell wall o r plasma membrane, such that the attachment o f the various binding proteins i s weakened (von Meyenburg, 1971). Inability t o grow at l o w lactose concentrations at 42" (Crandall and Koch, 1971).

Cannot f o r m functional transport systems f o r lactose o r uracil when growing at 42' (but glucose and a-methylglucoside transport are normal) (Crandall and Koch, 1971 ) . I t would be interesting t o have information about additional transport systems in these mutants,and also information about the presence o r absence o f k n o w n transport proteins (e.g., the M protein o f the lactose system).

Near lac (Nakamura, 1968). The relationship of acrA t o mrc (see below) has n o t been established

Sensitivity t o acriflavine (Nakamura, 1965).

General increase in binding (uptake?) o f basic dyes (acriflavine, toluidine blue, crystal violet, m e t h y l green, pyronine B ) and in sensitivity t o lipophilic substances (phenethyl alcohol, sodium dodecyl sulfate) (Nakamura, 1965,1966,1967,1968). Could result f r o m an alteration in the plasma membrane (Nakamura and Suganuma, 1972) or in the outer layers of the cell surface.

Near leu (Yura and Wada, 1968; Taylor. 19701.

Resistance t o azide o r phenethyl alcohol (Yura and Wada. 1968); formation o f filaments at 42O (Van de Putte eta/., 1964).

Transport properties have n o t been studied directly, b u t azi strains are thought t o have an altered plasma membrane (Vura and Wada, 1968).

Maps between leu and

Isolated as a mutation f r o m rough t o smooth colony mor. phology (Normark et a/., 1969).

Increased sensitivity to ampicillin, chloramphenicol, kanamycin, n o w biocin, actinomycin D, rifampicin, and gentian violet (Normark et al.. 1969; Normark, 1970) which,at least in the case o f gentian violet, can be correlated w i t h increased uptake (Normark and Westling, 1971 ). Believed t o be defective in the outer layer (LPS membrane) of the cell surface (Normark. 1970,1971).

ari, at 1.5 minutes (Normark, 1970).

~~

..

r

(Continuedl

TABLE 1 Mutations Affecting Membrane Transport (Continued) Multiple Transport Systems or General Membrane Permeability Organism

Membrane Property

Gene

Escherichia coli (Con‘t.I

IpcA. B

rn tcA

mtc8

tolAB

116

M u l t i p l e Transport Systems o r General Membrane Permeability Linkaqe

Method of lsolatinq Mutants

TransDort Defect

Near p y r F (Wijsman, 1972).

Isolated as temperaturesensitive mutants, able t o grow normally at 28" b u t not at 42"; 0.5% NaCl restores growth at the high ternperature (Wijsman, 1972).

Fail t o plasmolyze at 42", possibly because of a defective plasma m e m brane (Wijsman, 1972).

Between ara and lac (Tamaki eta/., 1971 1 .

Sensitivity t o novobiocin (Tamakietal., 1971).

Defect in t h e outer layer o f the cell surface (the LPS membrane), leading t o increased sensitivity t o novobiocin. spiramycin, and actinomycin D . and t o resistance t o some phages (Tamaki etal., 1971 1.

Between T6 and pur (Sugino, 1966); near lac (Otsuji, 1968); relationship t o acrA (see above) has not been established.

Sensitivity t o methylene blue (Sugino, 1966) o r t o mitomycin C (Otsuji, 1968; Imae. 1968.

Increased sensitivity t o a wide variety ot substances (mitomycin C, basic dyes, sodium dodecyl sulfate, sodium deoxycholate, colicins E l , E2, E3, K) (Sugino, 1966; Otsuji, 1968; Imae, 1968; Otsuji eta/., 1972).

Near metC; possibly identical w i t h to/C (see below) (Otsuji eta/., 1972).

Sensitivity t o mitomycin C (Otsuji e t a / . , 1972).

Resembles mtcA except that it is resistant t o colicin E l (Otsuji etal., 1972).

Near gal (Nomura and Witten, 1967; Nagel de Zwaig and Luria, 1967; H i l l and Holland 1967). Includes three complementat ion groups (Bernstein eta/., 1972).

Tolerance t o colicins E l , E2, E3, K, and A (Nomura and Witten, 1967; Nagel de Zwaig and Luria, 1967; Hill and Holland, 1967; Bernstein era/., 1971).

Between s t r a n d his (Nagel de Zwaig and Luria, 1967; Hill and Holland, 1967); near m e t C (Whitney, 1971).

Tolerance t o colicin E l (Nagel de Zwaig and Luria, 1967; Hill and Holland, 1967).

to/ mutants show increased sensitivity t o a variety of compounds: t o l A B t o deo xycholate, E D T A , vancomyci n, and bacitracin (Nagel de Zwaig and Luria, 1967; Bernstein eta/., 1971, 1972); tolC t o deoxycholate, methylene blue, and acridines (Nagel d e Zwaig and Luria, 1967); and t o l D t o various antibiotics and detergents (Rolfeetal., 1971). More recently, purified membranes f r o m t o l A 6 and to/C mutants have been demonstrated t o lack particular proteins found in wild-type membranes (Onodera eta/., 1970; R o l f e a n d Onodera. 1971).

Between s t r A and malQPT (Rolfe etal., 1971).

Tolerance t o colicins E2 and E3 at 40" (Nomura and Witten, 1967; Rolfe eta/., 1971). Sensitivity t o actinomycin D (Sekiguchi and lida, 1967).

117

Primary defect n o t known; the mutants show increased sensitivity t o lysozyme, as well as t o actinomycin D (Sekiguchi and lida. 1967).

(Continued)


Escherichia coli (Con%)

Membrane Property

Gene

-

M u l t i p l e Transport Systems or General Membrane Permeability Linkage -

Maps at 2 5 minutes (Taylor. 1 9 7 0 ) .


Transport Defect

Sensitivity t o synergistin A (Ennis, 1971).

Increased sensitivity t o a variety of drugs (strain H l O l : actinomycin D, bacitracin, carbomycin, clindamycin, erythromycin, spiramycin Ill, vernam y c i n A, and also t o detergents; strain H I S : acridine orange, amicetin, carbomycin, clindamycin, e r y t h romycin, fusidic acid, puromycin, spiromycin Ill,vernamycin). In addit i o n , HI35 is resistant t o phage T4. Primary defect n o t known.

A b i l i t y o f a tryptophan-requiring amber mutant t o grow in minimal medium supplemented w i t h t R N A f r o m an sul-carrying strain (Yamamoto eta/., 1971).

Increased uptake o f R N A (Yamamoto eta/., 1971).

Resistance t o phage TI; sensit i v i t y t o Cr3+ (Wang and Newton, 1969a).

May be a general defect in the plasma membrane, involving b o t h transport and the attachment o f phages and colicins. Wang and Newton (1969a.b) showed that t o n 8 mutants require very high concentrations o f Fe for growth, are defective in t h e 2 . 3 d i h y droxybenzoylserinedependent transp o r t o f Fe, and are abnormally sensitive t o Cr (which presumably inhibits the uptake of free Fe b y an alternative route; see discussion of Fe transp o r t in E. coli). More recently, Yanofsky (cited in Oxender. 1972a) has observed that deletions extending i n t o the t o n 6 region result i n decreased transport o f several amino acids. t o n 8 mutants are also resistant to phages TI and $80 and t o colicins €3. I,and V (Taylor, 1970).

Isolated, in a parent strain w i t h deletions covering mal8 (maltose permease) and l a c y (lactose permease), b y the ability t o grow on maltose and lactose. Alternatively, isolated in a wild-type parent strain b y increased sensitivity t o deoxycholate (Ricard era/., 1 9 7 0 ) .

Increased passive permeability t o sugars and increased sensitivity t o deoxycholate (Ricard e t a / . , 1970)

119

(Continued)

TABLE 1 Mutations Affecting Membrane Transport (Continuedl Multiple Transport Systems or General Membrane Permeability Organism

Membrane Property

Gene

Escherichia coli (Can't.)

Numerous mutants of E. coli have been isolated with primary defects in lipid synthesis, and some of them have been shown to possess secondary defects in transport or permeability.

-

plsA

fabA

fabB

120

Multiple Transport Systems o r General Membrane Permeability Linkage


Transport Defect

Isolated as a temperature-sensifive mutant; able t o grow at 30" b u t n o t at 41" (Hirota e r a / . . 1969).

Several membrane defects, including an abnormally large passive permeability t o o-nitrophenyl-p-D-ghlactoside (ONPG) (other compounds n o t tested), increased sensitivity t o deoxycholate, and intense fluorescence w i t h the d y e 1-anilino-8-naphthalenesulfonic acid (ANSI (Hirota e t a / . , 1969).

Requirement f o r glycerol (Hsu and Fox. 1970).

Primary defect in biosynthetic glycerol-3-phosphate dehydrogenase, which supplies L-glycerol 3-phosphate f o r lipid synthesis. When lipid synthesis is blocked b y withholding glycerol f r o m the growth medium, the p-galactoside transport system cannot be induced (Hsu and Fox, 1970).

Stimulation o f growth b y glycerol-3-phosphate ( K i t o e r a / . , 1969). Resistance t o radiation suicide b y glycerol 3-phosphate-H3 in a parent strain which incorporates exogenous glycerol 3-phosphate efficiently i n t o lipids; the parent strain IS defective in alkaline phosphatase and catabolic glycerol-3-phosphate dehydrogenase, and also constitutive for glycerol-3-phosphate transport (Cronan e t a / . , 1970; Godson, 1973).

Several mutants have been isolated w i t h defects in glycerol-3-phosphate acyltransferase, which is involved in the conversion o f glycerol 3-phosphate t o phosphatidic acid. One strain has an enzyme w i t h a 10-fold lower a f f i n i t y f o r glycerol 3phosphate ( K i t o er a/., 1969). and other strains have heatlabile enzymes (Cronan er a/., 1970; Hechemy and Goldfine, 1971; Godson, 1973).

Between p y r D and pyrC (Cronan e t a / . , 1 9 7 2 ) .

Growth requirement f o r an unsaturated f a t t y acid (Silbert and Vagelos. 1967; Cronan e t a / . , 1969; Henning et a / . , 1969; Esfahani et a/., 1969).

Primary defect in p-hydroxydecanoyl thioester dehydrase, the first enzyme in unsaturated fatty acid biosynthesis (Silbert and Vagelos, 1967; Esfahani e t a / . . 1969).

Between aroC and purF (Epstein and Fox, 1970; Schairer and Overath, 1969).

Same as above (Cronan e t a / . , 1969; Schairer and Overath, 1969; G. Wilson e r a / . , 1970).

Unable t o synthesize unsaturated f a t t y acids; enzymic defect n o t k n o w n (Cronan e t a / . , 1969; Birge and Vagelos, 1972). fabA and B mutants are useful for transport studies because their membrane composition can be varied according to the unsaturated f a t t y acid provided in the growth medium (Silbert and Vagelos, 1967; Silbert

Maps between purE and proC (Cronan and

Godson, 1972).

121

(Con tinued)


Membrane Property

Gene

Escherichia coli (Con %I


The car ( p t s ) mutations i n Salmonella, like the corresponding mutations in E. coli, are pleiotropic because they block the energy supply t o multiple carbohydrate transport systems (discussed under Mutations affecting the transport of carbohydrates).

Bacillus subtilis

Several mutants of B. subfilis are known in which lipid synthesis is defective; in some cases, corresponding defects in transport have been described.

122

-

Multiple Transport Systems or General Membrane Permeability Linkage


Transport Defect eta/., 1968; Silbert, 1970; Schairer and Overath, 1969; Overath e t a / . , 1970; Fox, 1969; Foxetal., 1970; G. Wilson eta/., 1970; Wilson and Fox, 1971; Esfahani et a/., 1969, 1971).

Requirement for high Mg concentrations for growth (Lusk era/., 1968). Now known to be inhibited b y Na; Mg overcomes the inhibition (Lusk and Kennedy, 1972).

Altered phospholipid metabolism, with Na stimulating the synthesis of cardiolipin and inhibiting t h e synthesis of phosphatidylethanolamine. Following the addition of Na, the P-galactoside transport system is also inhibited, suggesting a general change in the functioning of the membrane. Primary defect unknown (Lusk and Kennedy, 1972).

Primary defect in lipid synthesis, leading t o a deficiency in phosphotidylethanolamine (Beebe, 19711. Abnormally low rates of uptake of several amino acids (lysine, tryptophan, phenylalanine, serine, threonine, proline, methionine, glycine), pyruvic acid, and purines and pyrimidines (uracil, uridine, thymine, adenosine) (Beebe, 1972).

-

Requirement for glycerol (Mindich, 1970a).

Defective i n the synthesis of glycerol for lipids (probably via a glycerol-3phosphate dehydrogenase) (Mindich, 1970a). Used t o demonstrate that membrane proteins continue t o be made and that the citrate transport system can be induced in the absence of phospholipid synthesis (Mindich, 1970a.b; Willecke and Mindich, 1971).

Requirement for short branched-chain fatty acids (Willecke and Pardee, 1971b).

Defective i n branched-chain a-keto acid dehydrogenase (Willecke and Pardee, 1971b). ~ ~ _ _ _ _

123

(Continued)

TABLE 1 Mutations Affecting Membrane Transport (Continued) M u l t i p l e Transport Systems or General Membrane Permeability Organism

Membrane Property

Staph y-

Gene -

lococcus aureus

Neurospora crassa

A variety of Neurospora mutants has been isolated w i t h m u l t i p l e transport defects [see fpr-1, mod-5, nap, un(UMSOO),on (55701 t ) under Mutations affecting the transport of amino acids andpeptides1 ,as well as a series o f "osmotic" mutants that m a y have generalized defects in t h e cell membrane and/or cell wall.

In addition, t w o mutants of Neurospora are k n o w n t h a t are defective in f a t t y acid synthesis, but these strains have not been used f o r transport studies.

124

0s-1, 2 , 3, 4,5, cut, flm-2

cel (01)

Multiple Transport Systems or General Membrane Permeability Method of Isolating Mutants

Linkage

Transport Defect

Requirement for glycerol (Mindich, 1971).

Defective in the synthesis of glycerol for lipids. Used to demonstrate that the lactose transport system, induced in the absence of phospholipid synthesis, is only partly functional; lactose uptake by intact cellsdeclined to 30-50% of the control value, while phosphotransferase activity for pgalactosides in isolated membrane vesicles remained normal (Mindich, 1971).

os-l,3, 4, 5, cut, and f/m-2 all map on linkage group I, although not adjacent to one another (Mays, 1969). 0s-2 is on linkage group I V (Schroeder, cited in Mays, 1969).

Abnormal morphology (Kuwana, 1953; Perkins, 1959; Emerson, 1963). Inhibition of growth by the addition of 4% NaCl to the medium (Perkins, 1959; Emerson, 1963; Mays, 1969).

Primary defect unknown. These strains are inhibited in growth media of high osmolality (Perkins, 1959; Slayman and Slayman, 1965; Mays, 1969). are altered in the composition of their cell walls (Emerson and Emerson, 1958; Hamilton and Calvet, 1964; Trevethick and Metzenberg, 1966; Livingston, 1969). and have an abnormally high passive permeability to K and probably to other ions and small molecules (Slayman and Slayman, 1965). Both Mays (1969) and Slayman and Slayman (1965)have suggested that the primary change may be in some structural element of the cell membrane, such that the membrane becomes leaky and cell wall synthesis (which may be mediated by membranebound enzvmes) is abnormal.

Linkage group I V (Perkinseta/., 1962)

A segregant from a cross involving a morphological mutant isolated by S. R . Gross (Perkins eta/., 1962). Originally reported to grow on oleic acid, other higher fatty acids, or Tween, and named 01. More recently found to require saturated fatty acids and not t o grow on highly purified unsaturated fatty acids; renamed cel (chain elongation) (Henry and Keith, 1971).

Primary defect in the biosynthesis of long-chain saturated fatty acids.

Requirement for unsaturated fatty acids (Lein and Lein, 1949).

Primary defect in the synthesis of unsaturated fatty acids (Lein and Lein, 1949).

-

125

(Continued)


Membrane Property

Gene fas

Saccharomyces cerevisiae

01-1, 2, 3. 4

nys-1, 3, pol- 1 , 2 3. 5

Organism Man

Membrane Property The following five syndromes involve multiple abnormalities of renal tubular reabsorption. They may result from generalized damage t o the proximal tubule, as has been suggested in the case of Fanconi syndrome (Leaf, 1966). or from a metabolic disturbance in the supply of energy for transport (Rosenberg, 1969).

Disease Fanconi syndrome

Lowe's

syndrome

Busby syndrome

126

M u l t i p l e Transport Systems o r General Membrane Permeability Linkage


Transport Defect

Nine complementation groups (Schweizer eta/., 1971) involving at least three unlinked genetic regions (Henry and Fogel. 1971).

Requirement for long-chain f a t t y acids (Schweizer and Bolling, 1970; Schweizer e t a/., 1971; Henry and Fogel, 1971).

Defective in fatty acid synthetase, a muttienzyme complex o f molecular weight 2.3 x 106 containing seven enzymic activities and a carrier protein (Lynen, 1967; Schweizer and Bolling, 1970; Schweizer eta/., 1971; Henry and Fogel, 19711.

Four genes (Keith e t a / . , 1969). w i t h o/-1 mapping distal t o tr-5 o n chromosome VI I (Resnick and Mortimer,

Requirement for unsaturated f a t t y acids (Resnick and Mortimer, 1966).

Defective in the A9-desaturation o f palmitate and stearate ( K e i t h eta/., 1969; Wisnieski eta/., 19701.

Resistance t o polyene antibiotics, which b i n d t o sterols in the cell membrane (Ahmed and Woods, 1967; Woods, 1971; Molzahn and Woods,

Altered sterol composition (Woods, 1971; Thompson eta/., 1971; Molzahn and Woods, 1972). The relationship between nys mutants and 01 mutants (see above) has been discussed b y Bard

1972).

(1972).

1966). pol-1 and pol-3 are allelic t o nys-I and nys-3, respectively (Molzahn & Woods,

19721.

Mode of Inheritance

Abnormal Phenotype

Transport Defect

Can be inherited as an autosomal recessive (Dent and Harris, 1951). Can also be acquired f r o m poisoning w i t h heavy metals, Lysol, or outdated tetracycline, or as a secondary result o f Wilson’s disease, galactosemia, von Gierke’s disease, o r cystinosis (Leaf, 1966).

Increased excretion o f glucose and all amino acids, in spite o f normal or reduced plasma concentrations of these compounds; chronic acidosis; osteomalacia w i t h hypophosphaternia (reviewed in Leaf,

X-Linked recessive (Lowe eta/.. 1952)

Increased excretion o f glucose, amino acids, organic acids; decreased production o f ammonia in t h e kidney; glaucoma; cataracts; bone disease; mental retardation ( L o w e e t a/., 1952).

Primary defect unknown.

N o t established; the syndrome was observed in three of six children o f asymptomatic parents (Rowley er a/.,

Increased excretion o f all amino acids; growth retardation; poor muscular development; right ventricular hypertrophy (Rowley e t a/., 1961; Rosenbergetal., 19611.


1961).


19661.

(Continued] 127

128

CAROLYN W. SLAYMAN

TABLE 1 Mutations Affecting Membrane Transport (Continued) Multiple Transport Systems or General Membrane Permeability Orqanism Man (Con 't.I

Membrane Property

Disease G~UCOglycinuria

Glucoaminoaciduria

alanine transport system and presumably owed its resistance to the decreased uptake of D-cycloserine; the second had a normal transport system but increased activities of alanine racemase and D-alanine :D-alanine ligase, two enzymrs that are sensitive to inhibition by D-cycloserine. Similarly, BQchet et al. (1970) found that Saccharomyces cerevisiae could become resistant to canavanine (an arginine analog) in either of two ways: through mutations inactivating the arginine transport system, or through mutations (in three genetic loci) reducing the repressibility of ornithine transcarbamylase by arginine. For this reason analog-resistant mutants must always be tested directly for transport defects, as discussed below. In a variation of the analog resistance method, it is sometimes possible to find conditions under which naturally occurring compounds inhibit growth, so that the corresponding transport mutants can be selected by their failure to be inhibited. For example, the growth of wild-type E. coli I

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