INTERNATIONAL
REVIEW OF CYTOLOGY VOLUME92
Nuclear Genetics
ADVISORY EDITORS DONALD G. MURPHY H. W. BEAMS ROBERT G ...
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INTERNATIONAL
REVIEW OF CYTOLOGY VOLUME92
Nuclear Genetics
ADVISORY EDITORS DONALD G. MURPHY H. W. BEAMS ROBERT G . E. MURRAY HOWARD A. BERN RICHARD NOVICK GARY G. BORISY ANDREAS OKSCHE PIET BORST MURIEL J . ORD BHARAT B. CHATTOO STANLEY COHEN VLADIMIR R. PANTIC W. J. PEACOCK RENE COUTEAUX MARIE A. DIBERARDINO DARRYL C. REANNEY LIONEL I. REBHUN CHARLES J. FLICKINGER JEAN-PAUL REVEL OLUF GAMBORG M. NELLY GOLARZ DE BOURNE JOAN SMITH-SONNEBORN WILFRED STEIN YUKIO HIRAMOTO HEWSON SWIFT YUKINORI HIROTA K. TANAKA K. KUROSUMI DENNIS L. TAYLOR GIUSEPPE MILLONIG TADASHI UTAKOJI ARNOLD MITTELMAN ROY WIDDUS AUDREY MUGGLETON-HARRIS ALEXANDER YUDIN
INTERNATIONAL
Review of Cytology EDITED BY G. H. BOURNE
J . F. DANIELLI
St. George's University School of Medicine St. George's. Grenada
Danielli Associates Worcester, Massachusetts
West Indies
ASSISTANT EDITOR K. W. JEON Department of Zoology University o j Tennessee Knoxville. Tennessee
VOLUME92
Nuclear Genetics EDITED BY J . F. DANIELLI Danielli Associates Worcester, Massachusetts
ACADEMIC PRESS, INC. 1984 (Harcourt Brace Jovanovich, Publishers) Orlando San Diego New York London Toronto Montreal Sydney Tokyo
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LIBRARY OF CONGRESS CATALOG C A R D NUMBER: 52 - 52 03 I S B N 0-12-364492-5 PRINTED IN THE UNITED STATES OF AMERICA
84 85 86 87
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Contents CONTRIBUTORS
......................................
vii
Nitrate Assimilation in Eukaryotic Cells NIGELS . DUNN.COLEMAN. JOHNSMARRELLI. JR., AND REGINALD H . GARRET^
I. I1 . 111. IV . V.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biochemical Aspects of Nitrate Assimilation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetic Regulation of Nitrate Assimilation in Fungi . . . . . . . . . . . . . . . . . . . . . . . Genetics of Nitrate Assimilation in Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell and Molecular Biological Advances in Nitrate Assimilation . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1
2 12 26 39 45
Endocytosis and Exocytosis: Current Concepts of Vesicle Traffic in Animal Cells MARKC . WILLINGHAM A N D IRAPASTAN
I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Events at the Plasma Membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
111. IV . V. VI . VII .
Endocytic Vesicles: Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Kinetic Classes of Endocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Compartmentalization in the Golgi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Functions of Endocytic and Exocytic Membrane Traffic . . . . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
51 53 66 72 12
79
85 85
Stability of the Cellular Translation Process T. B . L. KIRKWOOD. R . HOLLIDAY. A N D R . F. ROSENRERGER I. I1. 111. IV . V. VI . VII . VIII . IX .
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Models of Error Propagation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Evolutionary Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nature of Proof or Disproof . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Measurement of Error Rates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Turnover of Proteins and Messages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Evidence from Prokaryotes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Evidence from Eukaryotes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
"
93 95 103 107 111
118 119 121 126 128
vi
CONTENTS
Chromosome and DNA-Mediated Gene Transfer in Cultured Mammalian Cells A . J . R . DE JONCEAND D . BOOTSMA
I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Technical Aspects of DMGT and CMGT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Uptake and Expression of Donor Genetic Materials in Recipient Cells . . . . . . . .
I1. 111. IV . V.
Applications of Gene Transfer in the Genetic Analysis of Mammalian Cells . . . . Conclusions and Future Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
133 135 139 147 154 156
DNA Methylation in Eukaryotic Cells AHARON RAZINA N D HOWARD CEDAR
I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. The Methylation Pattern of Eukaryotic DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. DNA Methylation and Gene Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
159 160 166 178 181
INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CONTENTS OF RECENTVOLUMES. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
187 191
Contributors Numbers in parentheses indicate the pages on which the authors' contributions begin.
D. BOOTSMA(133), Department of Cell Biology and Genetics, Erasmus University, 3000 DR Rotterdam, The Netherlands HOWARDCEDAR(1 59), Department of Cellular Biochemistry, The Hebrew University-Hadassah Medical School, Jerusalem 91010, Israel
A. J. R. DE J O N G E ~(133), Department of Cell Biology and Genetics, Erasmus University, 3000 DR Rotterdam, The Netherlands NIGELS. DUNN-COLEMAN (l), Central Research and Development Department, Experimental Station, E. I . DuPont Nemours and Co., Inc., Wilmington, Delaware I9898 REGINALDH. GARRETT( I ) , Department of Biology, University of Virginia, Charlottesville, Virginia 22901 R. HOLLIDAY (93), National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 I A A , England T. B. L. KIRKWOOD (93), National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 I A A , England IRA PASTAN(5l ) , Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20205 AHARONRAZIN (159), Department of Cellular Biochemistry, The Hebrew University-Hadassah Medical School, Jerusalem 91010, Israel R. F. ROSENBERGER(93), National Institute for Medical Research, The Ridgeway, Mill Hill, London M 7 IAA, England
'Present address: Department of Microbiology and Parasitology, Free University of Amsterdam, Medical Faculty, Amsterdam, The Netherlands. vii
...
Vlll
CONTRIBUTORS
JOHN SMARRELLI, JR.* (l), Department of Biology, University of Virginia, Charlottesville, Virginia 22901
MARKC. WILLINGHAM (5l), Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20205
2Present address: Department of Biology, Loyola University of Chicago, Chicago, Illinois 60626.
We regret to announce the death of Professor James F. Danielli on April 22, 1984, co-editor with Dr. Geoffrey H. Bourne of International Review of Cytology since publication of the first volume in 1952.
This Page Intentionally Left Blank
INTERNATIONAL REVIEW OF CYTOLOGY. VOL 92
Nitrate Assimilation in Eukaryotic Cells NIGELS. DUNN-COLEMAN,* JOHNSMARRELLI, JR., ? . I REGINALD H. GARRETT?
AND
*Central Research and Development Department Experimental Station, E.I. DuPont Nemours and Co. Inc., Wilmington, Delaware, and fDepartment of Biology, University of Virginia, Charlottesville, Virginia I
Introduction . . . . . . . . . . . Biochemical Aspects of Nitrate Assimilation. . . . . . . . . . . . . , . . . . . . A. General Features of Assimilatory Ni B. Fungal Nitrate Reductases . . . . . . . . C. Algal Nitrate Reductases.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Higher Plant Nitrate Reductases . . . . . . . . . . . . . . . . . . . . . . . . . . E. Assimilatory Nitrite Reductases . . . . , . . . . . . . . . . . . . . . . . . . . . F. Biochemical Regulation of Nitrate Assimilation . . . . . . . . . . . . . G. I n Vitro Complementation Studies on Nitrate Reductase . . . . . . 111. Genetic Regulation of Nitrate Assimilation in Fungi .... A. Genetic Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Autogenous Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Nitrogen Metabolism Repression of Nitrate Assimilation. . . . . . D. Possible Translational Control of Nitrate Assimilation . . . . . . . . E. Evidence for the Turnover of Nitrate Reductase . . . . . . . . . . . . . F. The Effect of Carbon Metabolism on Nitrate Assimilation. . . . . ____ IV. Genetics of Nitrate Assimilation in Plants . . . . . . . . . . .
I. 11.
.................................. V.
B . Conclusions .................................. Cell and Molecular Biological Advances in Nitrate Assimilation. . . . A. Somatic Hybridization Studies in Nitrate Assimilation . . . . . . . . B. Cloning of a Molybdenum Cofactor Gene from E . coli . . . . . . . C. Fungal Transformation Systems . . . . . . . . . . . . . . . . . . . . . . . . . . D. Plant Transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Cloning Nitrate Assimilation Genes-Concluding Remarks. . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2
6 6 7 9 11 12 12 18 19 23 25 25 26 26 38 39 39 41 42 43 43 45
I. Introduction Nitrate is the predominant form of combined nitrogen available within our oxidative environment, and its assimilation is achieved through its biological reduction to ammonium. The subsequent utilization of ammonium to form amino 'Present address: Department of Biology, Loyola University of Chicago, Chicago, Illinois 60626. 1 Copyright $8 1984 hy Academic Press. Inc All rights of reprodualon in any form reserved ISBN 0-12-364492-5
2
NIGEL S. DUNN-COLEMAN ET AL.
and amido-N compounds provides the link between the plethora of pathways of organic nitrogen metabolism and the pathways of inorganic nitrogen assimilation of which there are only two: nitrate assimilation and dinitrogen (N,) fixation. Losada and colleagues (Losada et al., 1981; Guerrero et al., 1981) have calculated, on the basis of relative carbon and nitrogen content of plants and the levels of CO, fixed by plants, that roughly 2 X lo4 megatons of inorganic nitrogen are assimilated annually. Since Bums and Hardy (1975) estimated that the overall chemical and biological fixation of dinitrogen was 2 X lo2 megatons, it follows that nitrate' assimilation exceeds nitrogen fixation by over 100-fold. Thus, the preponderance of nitrogen acquisition by the biosphere occurs via nitrate assimilation. Its significance to agriculture is enormous. The capability to assimilate nitrate is possessed by certain bacteria, some fungi, and virtually all algae and higher plants. It is absent from the animal kingdom. Nitrate assimilation represents a substantial energy expenditure by the cell when compared with ammonium utilization since eight reducing equivalents are consumed in the reduction of nitrate to ammonium. Consequently, cells which assimilate nitrate regulate this pathway to avoid wasteful use of reducing power when the end product, ammonium, is available. The form of regulation adopted varies in accordance with the metabolic pattern and status of the cell type but the fundamental purpose of the regulation is the same: to effect an economy of existence. For example, photosynthetic tissues assimilating nitrate show a rapid biochemical inactivation of this process when ammonium is presented. On the other hand, more rapidly proliferating fungal cells regulate through nitrate assimilation-specific gene expression. This review addresses the biochemistry, genetics, and regulation of nitrate assimilation in eukaryotic cells; particular emphasis is placed on the genetics of nitrate assimilation with an attentive eye to emerging molecular biological studies. Nitrate assimilation both invites the investigation of molecular biologists wishing to understand the regulation of metabolic potentialities in eukaryotic cells and intices manipulation by genetic engineers seeking to enhance plant productivity through application of recombinant DNA technology. Neither group has yet met fulfillment but the opportunities for both seem most promising.
11. Biochemical Aspects of Nitrate Assimilation
As indicated, the assimilation of nitrate is achieved through the eight-electron reduction of this oxidized inorganic anion, resulting in the formation of ammonium. This transformation requires two enzymatic steps, the two-electron reduction of nitrate to nitrite followed by the six-electron reduction of nitrite to ammonium:
NITRATE ASSIMILATION IN EUKARYOTIC CELLS
NO?
2e
~
niuarc reductase
3
6r -
NOT -N H$ nitme rcduclaw
The enzymes mediating these steps are nitrate reductase (EC 1.6.6.1-3) and nitrite reductase (EC 1.6.6.4 and 1.7.7. l ) , respectively. OF ASSIMILATORY NITRATEREDUCTASES A, GENERALFEATURES
The biochemical aspects of assimilatory nitrate reductases have been extensively reviewed recently (Garrett and Amy, 1978; Hewitt and Notton, 1980; Beevers and Hageman, 1980; Guerrero et al., 1981; Vennesland and Guerrero, 1979; Losada et al., 1981). This section will initially focus on features shared by all assimilatory nitrate reductases and subsequently detail unique characteristics of the enzyme from algal, fungal, and higher plant sources. Assimilatory nitrate reductases are soluble, electron-transferring proteins, 200,000-300,000 in molecular weight. Electron transfer is generally regarded to be mediated by enzyme-bound heme iron, flavin adenine dinucleotide (FAD), and molybdenum cofactor. These components function as electron carriers between the physically separated pyridine nucleotide oxidation site and nitrate reduction site (Campbell and Smarrelli, 1978, 1983). The physiological activity involves the reduction of nitrate to nitrite occurring at the molybdenum site, with reducing power generated by pyridine nucleotides donated to the catalyst probably via a specific enzyme sulfhydryl group (Amy et al.. 1977). In addition to this physiological activity, apparent nonphysiological activities can be shown in vitro. The first is termed a dehydrogenase (diaphorase) activity in which nitrate reductase can mediate the pyridine nucleotide-linked reduction of one- or twoelectron acceptors such as ferricyanide, cytochrome c, or dichlorophenolindophenol. The molybdenum moiety is not involved in these reactions. The second type of activity involves the reduction of nitrate with reducing power generated either by reduced flavins or viologen dyes. The pyridine nucleotide reduction site is not involved in this reductase activity and electrons are added probably to either heme or molybdenum cofactor. The study of these partial activities has provided valuable insights into the overall nature of the enzyme since the characteristic response of these activities reflect intrinsic properties of the nitrate reductase. For example, these reactions have been found to be differentially inhibited. Sulfhydryl binding agents such as p-hydroxymercuribenzoate inhibit both the pyridine nucleotide-linked nitrate reductase and dehydrogenase reactions (Garrett and Nason, 1969; Schrader et al., 1968). Metal-binding agents such as cyanide inhibit only the reactions involving the molybdenum moiety, while having no influence over the dehydrogenase activity of nitrate reductase (Garrett and Nason, 1969; Hewitt and Notton, 1980).
4
NIGEL S . DUNN-COLEMAN ET AL.
Further, polyvalent monospecific antisera against nitrate reductase differentially inhibit the partial activities (Amy and Garrett, 1979; Funkhouser and Ramadoss, 1980; Smarrelli and Campbell, 1981). From these and similar studies, the general features of the typical assimilatory nitrate reductase have been revealed, as depicted in the following scheme: MVH
L
NADPH + [FAD + cytochrome-bsY7+ (Mo)] + N O 1 I cytochrome c , Fe3(CN),
Additional information and analogy provides a more complete description of overall electron flow from the binding and oxidation of reduced pyridine nucleotide to the terminal reduction of nitrate to nitrite. Pyridine nucleotides are thought to bind to a supersecondary structure of the enzyme called the dinucleotide fold (Solomonson, 1975). Dinucleotide folds are typically composed of about 120 amino acid residues in five or six parallel strands to form a P-sheet core, the strands being connected by a-helical intrastrand loops located above and below the P-sheet (Rossman et al., 1974). Following A-side oxidation of the reduced pyridine nucleotide (Guerrero et al., 1977), the flavin region of the enzyme becomes reduced. Flavin adenine dinucleotide appears noncovalently bound to nitrate reductase. The FAD is easily dissociable from the enzyme from Neurospora crassa (Garrett and Nason, 1967), while showing very tight binding in other assimilatory nitrate reductases. Amy et nl. (1977) described an important sulfhydryl group which apparently mediates electron transfer between NADPH and FAD. From FAD, electrons are subsequently shuttled to a 6-type cytochrome. First identified by Garrett and Nason (1967) for the enzyme from N . crassa, and subsequently confirmed for other nitrate reductases (Solomonson et al., 1975; Guerrero and Gutierrez, 1977; Notton et al., 1977; De la Rosa et al., 1981; Minagawa and Yoshimoto, 1982), it has been termed cytochrome bSs7 since it displays an a-peak at 557 nm in a reduced versus oxidized difference spectrum. The terminal electron acceptor of the nitrate reductase protein is thought to be the molybdenum cofactor. Molybdenum cofactor was once regarded as a proteinaceous component (Nason et d . ,1970). However, more recent studies reveal that it is most likely a low-molecular-weight, molybdenum-binding, urothionelike molecule termed molybdopterin (Johnson et al., 1980; Johnson and Rajagopalan, 1982). The actual oxidation states of molybdenum during nitrate reduction have not been established unambiguously (Jacob and Orme-Johnson, 1980). Although many of the biochemical features of assimilatory nitrate reductases appear well established, the actual structure and role of the components involved in electron flow are only poorly understood. Elucidation of these aspects, however, involves the use of large quantities of protein, a formidable task given the low
NITRATE ASSIMILATION IN EUKARYOTIC CELLS
5
cellular concentration and relative instability of all nitrate reductases. The low cellular concentration of this enzyme is attributed to its remarkable catalytic efficiency; its turnover number or molecular activity is 18,000 (as a point of comparison, the molecular activity of succinate dehydrogenase is 1200) (Garrett and Amy, 1978). B. FUNGAL NITRATE REDUCTASES
The best characterized fungal nitrate reductases are those derived from Neurosporu crussa (Garrett and Nason, 1969; Pan and Nason, 1978; Homer, 1983), Penicillium chrysogenum (Renosto et al., 198l), Rhodotorula glutinis (Guerrero and Gutierrez, 1977), and Aspergillus nidulans (Minagawa and Yoshimoto, 1982). Table I summarizes several of the important physical properties of these enzymes. With the exception of the enzyme from A . nidulans (see also Downey and Steiner, 1979), all appear to be homodimers, with molecular weights in excess of 200,000. All are specific for NADPH as pyridine nucleotide electron donor, display pH optima of approximately 7.5, and possess an easily dissociable FAD. All apparently homogeneous enzyme preparations except that from A . nidulans display specific activities greater than 100 unitslmg protein (1 unit being 1 kmol of nitrate reduced per minute). The best preparations of these fungal nitrate reductases were obtained using affinity chromatography (FAD-
TABLE I FUNGAL NITRATEREDUCTASES AND HIGHER PLANTNADH-NITRATE REDUCTASES
Source
Native molecular weight ( X 103)
Subunit molecular weight ( X lo3)
Reference
Fungi
N . crassa
230
N . crassa P . chrysogenum R . glutinis A . nidulans A . nidulans
290 200 230 180
Higher plants Squash Barley Tobacco Spinach
-
230 220 220 190
115 145
100 118 59 + 38 90
115 110 110
120 (major)
Garrett and Nason (1969); Pan and Nason ( 1978) Homer (1983) Renosto e t a l . (1981) Guerrero and Gutierrez (1977) Minagawa and Yoshimoto (1982) Tomsett (1983, personal communication)
Redinbaugh and Campbell (1983) Kuo ef a / . (1982) Mendel and Miiller (1980) Notton and Hewitt (1979)
6
NIGEL S . DUNN-COLEMAN ET AL
Sepharose or Blue Dextran-Sepharose) in combination with conventional techniques. C. ALGALNITRATEREDUCTASES The best characterized algal nitrate reductases are those from Chlorella vulgaris (Solomonson et al., 1974, 1975; Giri and Ramadoss, 1979; Howard and Solomonson, 1982) and Ankisfrodesmus braunii (De la Rosa, 1981). However, the literature reveals discrepancies regarding the subunit composition of the enzyme from Chlorella. Initial reports (Solomonson et al., 1975) described the Chlorella vulgaris nitrate reductase as a trimer with a native molecular weight of 356,000. Later studies (Giri and Ramadoss, 1979) concluded the enzyme to be a trimer of molecular weight 280,000. The most recent data (Howard and Solomonson, 1982) conclude that the Chlorella nitrate reductase is a homotetramer with a molecular weight of 360,000. The nitrate reductase from Ankistrodesmus braunii has been described as an octamer with a molecular weight of 370,000. In other significant features, pH optima, pyridine nucleotide specificity, prosthetic group involvement, and molecular activity, these two representative green algal nitrate reductases are not significantly different from each other or from fungal nitrate reductases. FAD is not easily dissociable from either algal enzyme, in contrast to the situation found in fungal nitrate reductases. D. HIGHERPLANTNITRATEREDUCTASES Immunological data suggest a degree of similarity between algal, fungal, and plant enzymes (Smarrelli and Campbell, 1981), but structural and catalytic diversity of the enzyme exists even within a single plant. However, the basis of this diversity is obscured by the difficulty in obtaining a protein unmodified by the purification procedures. Thus, discerning intrinsic differences is problematical. Well characterized enzyme has been obtained from corn, barley, wheat, spinach, squash, and tobacco (Campbell and Smarrelli, 1978; Redinbaugh and Campbell, 1983; Kuo et al., 1982; Sherrard and Dalling, 1979; Notton et al., 1977; Mendel and Muller, 1980). There is not only a diversity of structure between the well characterized leaf nitrate reductases, but also different enzymes in the same tissue (e.g., soybean leaves) and tissue-specific enzymes. Typically these enzymes have been distinguished with some reliability by their pyridine nucleotide specificity (e.g., corn leaves and roots). The majority of leaf nitrate reductases are highly specific for NADH. The physical properties of representatives of this group are also shown in Table I . This group of higher plant enzymes can thus be characterized as homodimeric with subunit molecular weights of approximately 115,000. All leaves thus far examined apparently contain this type of nitrate reductase, except those from Erythrina senegalensis (Stewart and Orebamjo,
NITRATE ASSIMILATION IN EUKARYOTIC CELLS
7
1979). This tropical legume contains an enzyme which can accept electrons from either NADH or NADPH. The bispecific NAD(P)H-nitrate reductase has been described in three general cases. First, it can accompany the typical NADH-nitrate reductase in the same tissue. This pattern was found in soybean leaves and cotyledons (Jolly et al., 1976; Orihuel-Iranzo and Campbell, 1980). In rice seedlings, the two enzymes respond to different induction conditions (Shen et al., 1976). Despite their pyridine nucleotide specificity differences, the presence of two nitrate reductases in one tissue has not yet been adequately explained. The second case of the bispecific nitrate reductase was reported for corn roots and scutella (Redinbaugh and Campbell, 1981; Campbell, 1978). Since corn leaves contain only the typical NADH-nitrate reductase, there appears to be tissue-specific control over synthesis of the bispecific enzyme. The third case is the synthesis of the bispecific enzyme in response to mutation of the NADH-nitrate reductase (Dailey et al., 1982a). Mutant barley seedlings which lack NADH-nitrate reductase activity (nar-la) have been found to contain a bispecific nitrate reductase which is absent from normal tissue. Thus, multiple genes for nitrate reductases apparently exist in higher plants. The total purification of higher plant nitrate reductase has required the application of affinity chromatography, the most prevalent matrix being blue-Sepharose. Rapid purification is essential since numerous higher plant proteases have been identified (Hageman and Reed, 1979). Proteinase inhibitors have been somewhat effective with leupeptin being the best inhibitor (Wray and Kirk, 1981). E. ASSIMILATORY NITRITEREDUCTASES Nitrite reductase, the second enzyme of the nitrate assimilatory pathway, catalyzes the six-electron reduction of nitrite to ammonium. The reduction proceeds as follows, apparently with no intermediates released in the reaction NO,
+
6e-
+ 8H+
+
NHa
+ 2H20
Although catalyzing the same reaction, the nitrite reductases from photosynthetic organisms (EC 1.7.7.1) are significantly different from those from nonphotosynthetic sources (EC 1.6.6.4), particularly in molecular weight and electron donor specificity. Nitrite reductases from several photosynthetic sources, including Chlorella fusca, spinach, squash, and Porphyra yezornsis, have been purified to homogeneity (Vennesland and Guerrero, 1979, and references therein). The nitrite reductase from the fungus N . crassa also has been purified to homogeneity (Greenbaum et al., 1978; Prodouz and Garrett, 1981). The two best characterized nitrite reductases have been obtained from spinach leaves (Vega and Kamin, 1977; Lancaster et al., 1979) and N . crassa (Garrett, 1978; Prodouz
8
NIGEL S. DUNN-COLEMAN ET AL.
and Garrett, 1981) and will be discussed as representative examples to compare nitrite reductases from photosynthetic and nonphotosynthetic organisms. Assimilatory nitrite reductases have in common the possession of iron-sulfur center and siroheme prosthetic groups. The iron-sulfur center in spinach nitrite reductase is organized as a tetranuclear Fe,S, cluster (Lancaster et al., 1979); the Neurospora nitrite reductase is thought to contain two such Fe,S, centers (Prodouz and Garrett, 1981). Siroheme is an iron tetrahydroporphyrin of the isobacteriochlorin type, containing eight carboxyl groups and having two adjacent pyrole rings reduced (Scott et al., 1978). Siroheme is also found in assimilatory sulfite reductase (Siege1 et al., 1974). The siroheme function likely serves as the site of binding and reduction of nitrite in Neurospora (Vega et al., 1975; Garrett, 1978) and spinach (Vega and Kamin, 1977). Siegel’s laboratory (Wilkerson et al., 1983) has evidence from Mossbauer spectroscopic studies that the Fe,S, cluster and the siroheme of spinach nitrite reductase undergo exchange interactions, indicating that these two centers are chemically linked. It thus seems probable that the nitrite-reducing center of assimilatory nitrite reductases is the chemically coupled Fe,S,/siroheme prosthetic pair: 6e-
4
[Fe4S4/siroherne]
G
NO, NHJ
+ 8H+ + 2H20
This basic nitrite-reducing unit is found in association with a 61,000-MW protein in photosynthetic organisms where ferredoxin serves as the electron donor (Vega and Karmin, 1977). In contrast, the enzyme from nonphotosynthetic organisms as typified by Neurospora C ~ U S S Uis a 290,000-MW homodimeric flavoprotein of 140,000-MW subunits which utilizes reduced pyridine nucleotide (either NADPH or NADH) as electron donor (Lafferty and Garrett, 1974; Greenbaum et al., 1978; Prodouz and Garrett, 1981). Thus, the following electron transfer schemes aptly represent the nitrite reductases of photosynthetic and nonphotosynthetic cells: Spinach.
6 Fdred-+ [Fe4S4/siroheme]+ NO,
Neurospora
3NAD(P)H + [FAD -+ Fe4S4/siroheme]
--f
NO?
It is interesting to note that nonphotosynthetic cells apparently require a relatively larger and more complex nitrite reductase in order to utilize the reduced pyridine nucleotides as electron donors since they lack ferrodoxin and the photochemical means to reduce it (Garrett, 1978). The comparative properties of spinach and Neurospora nitrite reductase are summarized in Table 11. Both classes of enzyme are also inhibited by sulfhydryl agents such as pHMB, the anions CN- and sulfite which are substrate analogs, and CO which binds avidly to nitrite reductase in which the siroheme moiety is reduced (Greenbaum et al., 1978). The Neurosporu nitrite reductase, like the nitrate reductase in this organism,
NITRATE ASSIMILATION IN EUKARYOTIC CELLS
9
TABLE I1 COMPARATIVE PROPERTIES OF ASSIMILATORY N I T R I TREDUCTAS~S ~ Property Molecular weight Subunits Subunit molecular weight Electron donor Flavoprotein Iron-sulfur clusters Siroheme Specific activity (+ma1 NO,
Spinacho 61,000 1
-
reducedimg protein)
61,000 Ferredoxin No One Fe4S4 Yes 108
N . crassab
290,000 2 (a2-type homodimer) 140,000 NAD(P)H Yes (FAD) Two Fe4S4 Yes 27
“Vega and Kamin (1977) and Lancaster el al. (1979). bGreenbaum et al. (1978) and Prodouz and Garrett (1981).
has the capacity to catalyze a number of partial electron transfer activities in vitro. These include a FAD-dependent NAD(P)H-diaphorase activity for which cytochrome c, ferricyanide, or dichlorophenol indophenol serve as electron acceptor, and a FAD-independent dithionite-nitrite reductase activity (Lafferty and Garrett, 1974; Vega, 1976). Further this enzyme can catalyze the two-electron reduction of hydroxylamine to ammonium in a reaction using NAD(P)H and requiring FAD. This NADPH-hydroxylamine reductase activity has no physiological significance because of the high K , for NH,OH, namely 3 mM.
F. BIOCHEMICAL REGULATION OF NITRATEASSIMILATION As previously mentioned, nitrate assimilation is an energetically expensive process, requiring four equivalents of reduced pyridine nucleotide per nitrate reduced. Consequently, regulation of this pathway, particularly by inactivation when sufficient ammonium levels are available, would be economically advantageous. Further, the efficient site of regulation would be the first step, nitrate reductase, effectively halting assimilation and preventing accumulation of the toxic metabolite, nitrite. Ammonium is not a direct feedback inhibitor of nitrate assimilation, nor are any of the primary amino compounds such as glutamine or glutamate. However, biochemical regulation of nitrate reductase activity through oxidation-reduction interconversion of the enzyme has been suggested (Losada, 1974). Losada demonstrated that algal and higher plant nitrate reductases could be inactivated in vitro by preincubation with reductants such as NADH or dithionite and then reactivated by oxidants such as ferricyanide. Losada suggested nitrate reductase could therefore exist in two interconvertible forms, an oxidized, active form and a reduced, inactive species. The generality of this mechanism is not universal; the N . crclssa nitrate reductase is not subject to reductive inactiva-
10
NlGEL S. DUNN-COLEMAN ET AL.
tion in vivo. Using purified nitrate reductase from Chlorella vulgaris,Lorimer et al. (1974) demonstrated that two components were required for rapid inactivation of nitrate reductase, NADH and cyanide. Cyanide reacted stoichiometrically with NADH-reduced enzyme to give a stable enzyme-cyanide complex having an association constant to 1Olo M . Further, stoichiometric amounts of cyanide were released when physiologically inactivated nitrate reductase, isolated from cells exposed to ammonium, was reactivated. This observation suggested a physiological role for cyanide in regulating nitrate reductase activity. These observations were expanded into a model for the metabolic regulation of nitrate assimilation (Solomonson and Spehar, 1977) in which cyanide was recognized as the simplest carbon-nitrogen compound and postulated to be pivotal in integrating and regulating carbon and nitrogen assimilation. Cyanide in vivo was hypothesized to arise from hydroxylamine and glyoxylate, hydroxylamine in turn being an intermediate product of nitrite reduction by nitrite reductase. The origin of CN- remains unclear and thus the model, though attractive, remains speculative. It is clear however that nitrate reductase is present in an inactive, cyanide-bound state in vivo in algal cells treated with ammonium or in nitrategrown cells in late log phase. Several high-molecular-weight inhibitors have been reported to affect higher plant nitrate reductases. These inhibitors can be classified as either proteolytic enzymes or binding proteins. Nitrate reductase proteases have been implicated in wheat leaves and maize roots (Wallace, 1974, 1975; Sherrard et al., 1979; Yamaya et al., 1980). Both inhibitors were found to be heat and EDTA sensitive. Other reports have implicated two nitrate reductase inactivators in N . c r a m (Walls et al., 1978; Horner, 1983). These inactivators which were apparently proteolytic and could be inhibited by EDTA and/or phenylmethylsulfonyl fluoride may be involved in the turnover of nitrate reductase (Section 111,E). Inhibitors which bind nitrate reductase have been reported for a number of plant species (Yamaya et al., 1980, and references therein; Jolly and Tolbert, 1978). Although proteases and other proteinaceous inhibitors do affect nitrate reductase activity, their functions remain generally uncharacterized and their roles in the regulation of physiological nitrate reductase activity thus are unclear. These agents increase the difficulty in obtaining an unaltered nitrate reductase protein during purification and thereby complicate the characterization of the native enzyme form. Reversible inactivation of nitrate reductase from higher plants, algae, or fungi has been achieved by either dialysis or gel filtration, with reactivation possible by EDTA or various amino acids (Smarrelli and Campbell, 1983; Ketchum et al., 1977). The biological significance of these effects is uncertain. In addition, higher plant nitrate reductase was found to have an affinity for heavy metals (Smarrelli and Campbell, 1983; Nason and Evans, 1953). However, this inhibi-
NITRATE ASSIMILATION IN EUKARYOTIC CELLS
11
tion-reactivation is unlikely to have physiological significance and is important only in the handling of nitrate reductase in vitro.
G. In Vitro COMPLEMENTATION STUDIESON NITRATEREDUCTASE In 1970, Nason and his collaborators (Nason et al., 1970) reported that NADPH-nitrate reductase activity could be reconstituted by combining extracts of a particular nitrate reductase-deficient mutant strain, nit-1 , with extracts of various other nit mutants strains or wild-type extracts. Subsequent experimentation (Ketchum et al., 1970; Nason et al., 1971) gave evidence that a molybdenum-containing component or molybdenum cofactor was provided by the wild-type or other extract added to the nit-1 extract, and this endogenous molybdenum cofactor complemented apo-nitrate reductase in the nit- 1 to form active NADPH-nitrate reductase molecules. These observations essentially gave birth to research on the nature of the molybdenum cofactor and the biological role of molybdenum, a rapidly expanding area of inorganic biochemistry beyond the scope of this review. Nevertheless, these observations had important bearing on our understanding of the structure and function of nitrate reductase. The nit-1 mutant contains a NADPH-cytochrome c reductase (diaphorase) activity displaying a sedimentation coefficient of 4.5 (4.5 S). This activity is increased in nit-1 mycelia grown on nitrate (Nason et al., 1970) and is cross-reactive to antinitrate reductase antibodies (Amy and Garrett, 1979). The conclusion is this NADPH-cytochrome c reductase is apo-nitrate reductase, i.e., molybdenumfree enzyme subunits, and that in the presence of a low-molecular-weight (< 1000) molybdenum-containing cofactor (molybdopterin), these apoprotein subunits dimerize to yield native NADPH-nitrate reductase (8 s). Further, it has recently been confirmed that the reconstructed enzyme exhibits properties similar to the wild-type enzyme (Homer, 1983). The in vitro complementation reaction has been instrumental in elucidating the nature of molybdenum cofactor and researchers have also utilized it as a means to characterize mutants deficient in molybdenum cofactor or apoprotein. Acidified xanthine oxidase is generally used as a source of molybdenum cofactor (Tachiki and Nason, 1983), while extracts of nit-1 are utilized as the source of apo-nitrate reductase to test for the presence of active molybdenum cofactor (for example, see Mendel et al., 1981, 1982a,b). Further, high concentrations of molybdate have been shown to restore the activity of some molybdenum cofactor mutants (Tachiki and Nason, 1983; Mendel et al., 1981). Experiments analogous to those described for N . crassa have been done with niaD and c m mutants from Aspergillus nidulans, again resulting in the formation of a functional nitrate reductase (Garrett and Cove, 1976). In vitro reconstruction of active nitrate reductase has also been performed with
12
NIGEL S. DUNN-COLEMAN ET AL
higher plants. Active nitrate reductase from molybdenum-deficient spinach leaves could be obtained by addition of acid-treated spinach nitrate reductase (Rucklidge et al., 1976) and, as cited, cnx-type mutants in tobacco were reconstituted by acid-treated xanthine oxidase (Mendel et al., 1981, 1982a,b), suggesting that they are defective in molybenum cofactor. Recently, barley mutants were analyzed by reconstitution experiments using spinach apoenzyme or molybdenum cofactor (Notton et al., 1983).
111. Genetic Regulation of Nitrate Assimilation in Fungi The regulation of nitrate assimilation in fungi occurs predominantly at the level of gene expression. In the presence of nitrate or nitrite, the enzymes of nitrate assimilation are induced, provided ammonium is absent. In the presence of ammonium, or reduced nitrogenous metabolites such as glutamine, nitrate assimilation is repressed even if nitrate is available. Thus, both induction and repression are operant here. These conclusions are based on the genetic analysis described below. A. GENETICANALYSIS 1. Aspergillus nidulans
A great deal of the understanding about the genetics and the biochemistry of nitrate assimilation in eukaryotic organisms has come from the study of this pathway in the ascomycete fungi Aspergillus nidulans and Neurospora crassa. For example, Birkett and Rowlands (1981) have recently undertaken a genetic analysis of nitrate assimilation in Penicillium chrysogerzum and shown it to be identical to that of A. nidulans (see Table V). Pateman and Cove (Cove and Pateman, 1963, 1969; Pateman et al., 1964; Pateman and Cove, 1967) initially screened A. nidulans for strains unable to utilize nitrate as a nitrogen source. These experiments led to the isolation of both structural and regulatory mutants of nitrate assimilation (Table 111). For example, niaD mutants although unable to grow on nitrate could utilize nitrite as a nitrogen source. MacDonald and Cove (1974) isolated several temperature-sensitive niaD mutants and showed that their nitrate reductase activity was thermolabile. These results substantiated the view that the niaD locus encodes the nitrate reductase apoprotein. A second class of mutants isolated, niiA, were unable to utilize nitrate or nitrite as the nitrogen source. These niiA mutants lacked nitrite reductase activity and the niiA locus is believed to be the structural gene for nitrite reductase (Pateman et al., 1964). A third class of mutants were unable to utilize nitrate or hypoxanthine as a nitrogen source since they lacked both nitrate reductase and xanthine dehydrogenase
TABLE TABLE111 111 FUNGAL NITRATE FUNGAL NITRATEASSIMILAIIUN ASSIMILATIONMUTANTS MUTANTS A.A niduluns . .crussu . nidulans N N crassa P .P.chwsogenum chwsogenum niaD niaD
nir-3 nit-3
niuA niaA
Nitrate reductare apnprotein structural gene
niiA niiA nirA nirA
nit-6 nit-6 nir-4/5 nit-4/5
niiA niiA nlrA nirA
areA areA
nit-2 nit-2
--
-
nit-1 nit- 1 nit-9ABC nit-9ABC
cmABC cmABC
Nitrite reductase apoprotein structural gene trans-acting reguhtoiy gene required for the expression of nitrate reductase and nitrite reductase under conditions of induction by nitrate trans-acting regulatory gene required for the expression of a wide range of ammonium-generating enzymes, including nitrate reductase and nitrite reductase Encodes Encodesananenzyme enzymefor forMoCo MoCosynthesis? synthesis? Possibly Possiblya asingle singlegene genewith with3 3intracistronic intracistroniccomplementation complementation groups contiguousgenes genescnxA cnxAand andcnxC, cnxC,with withcnxB CUB groupsoror2 2contiguous mutants mutantsbeing beinga adouble doubleloss lossclass class Encodes Encodesananinzyme enzymefor forMoCo MoCosynthesis? synthesis? cnxE CUEmutants mutantsarc aremolybdatc molybdatercpairable repairableand andmay maybebedcfective defective inininscnion insertionofofInolybdate molybdateinto intothc thecofactor cofactor
cmABC cmABC
-
-
I
I
cnrE clUE
-
-
cnxF cnxF
-
UL TH clUH
cnxG CnxG cnxJ cmJ
~~
cnxD cnxD cnxE cnxE
ClZIF” cnxF”
--
C fCI XU GU CP
nil-7 nit-7 nit-8 nit4
--
-
crnA crnA
-
-
-
---
~
Function Function
~
--
Encodes Encodesenzyme enzymefor forMoCo MoCobiosynthesis? biosynthesis? Structural MoCo? Structuralcomponent componentofofMoCo? Encodes Encodesananenzyme enzymefor forMoCo MoCobiosynthesis? biosynthesis? Possible regulatory locus, affecting MoCo levels It is not certain to which A. nidulans c m loci nit-7 and n i t 4 correspond Nitrate permease locus
ReTerence Reference
of N . c r a m , see Tomsett and Garrett (1980, 19x1) For reviews of A . nidulans, see Cove (1976a.b, 1979) and Tonisett and Cove ( 1979) Fnr reviews
P . ckqsogenum work by Birkett and Rowlands (1981) 3
I
+
Molybdenum cofactor (MoCo) loci. Mutations Mutationsininany anyone oneol’ ofthcsc theselac1 loci leads leadstotoaaloss lossofofnitratc nitratercductasc, reductase, purine purinehydrvxylase hydroxylase1,I ,and andpurine purine hydmxylasc activity hydroxylaseI1I1activity
I
Arst et al. ( I 982a)
Tonisett and Cove (1979); Brownlee and Arst 11983)
~
aIt is isnot notknown knownif ifthe thenomenclature nomenclatureused usedwith withthe theP.P.chtysogenum chrysogenumcn.r cnxmutants mutantsisisstrictly strictlyequivalent equivalenttotothe thecnrF cnxFand andcnxG cnxGmutants mutantsofofAA. . niduluns. nidulans.
14
NIGEL S. DUNN-COLEMAN ET AL
(XDH) activity (XDH activity in A . nidulans resides in two molybdoenzymes, called purine hydroxylases I and 11) (Pateman el al., 1964). These mutants were designed as cnx, indicating that they were defective in the biosynthesis of a cofactor needed for both nitrate reductase and xanthine dehydrogenase activity. It was found that these mutants could be further classified into seven complementation groups (cmABC, cnxE, c m F , c m H , and c m G ) at five genetic loci, the cmABC locus being possibly a single gene with three intracistronic complementation groups or two contigous genes, cnxA and cnxC, with cmB mutants being a double loss class. Arst et al. (1970) demonstrated that mutants at the cnxE locus were molybdate repairable, since high concentrations of exogenous molybdate partially restored both nitrate reductase and xanthine dehydrogenase activity. Similar molybdate-repairable mutants have been found in P . chrysogenum (Birkett and Rowlands, 1981) and in higher plants such as Nicotiana tabacum (Mendel and Muller, 1979). MacDonald and Cove (1974) have isolated temperature-sensitive alleles of cnxE, cnxF, and cnxH, but only cnxH temperaturesensitive mutants have a thermolabile nitrate reductase activity. They tentatively concluded that the cnxH gene product may have a structural role in the nitrate reductase molecule. However, the function of any cnx locus is presently unknown; it is thought that they encode for enzymes concerned with molybdenum acquisition and molybdenum cofactor biosynthesis. Arst et a f . (19824 isolated mutants at a sixth locus, cmJ. Two allelic mutants, cnxJl and 52, have reduced molybdenum cofactor levels, rendering these mutants more sensitive to toxic analogs such as methylammonium when grown on nitrate. They speculated that the cmJ locus has a regulatory role in molybdenum cofactor regulation. They also reported the isolation of cryosensitive cnxC mutants unable to utilize nitrate or hypoxanthine as a nitrogen source at 25°C but able to grow at 37°C. On this basis Arst et al. (1982a) suggested that the c r d gene product may also have a structural role in the molybdenum cofactor. Garrett and Cove (1976) undertook a biochemical study of the molybdenum cofactor mutants ofA. nidulans by in vitro complementation (see Section I1,G). They found that cohomogenization of mycelia from the niaD26 structural gene deletion mutant with mycelia from either cnxA6, cnxE29, cnxF12, cnnG4, or cmH3 resulted in the formation of NADPHnitrate reductase activity. Using this in vitro complementation of nitrate reductase activity to study the regulation of molybdenum cofactor synthesis, it was found that more molybdenum cofactor was present in mycelia grown with nitrate as a nitrogen source than with ammonium. Further, the results with niaD; nirA- , and niuD; nirAc double mutants indicated that the nirA gene (see Section II1,B) did not play a role in the regulation of molybdenum cofactor snythesis. Many mutations in the cnx and niaD genes lead to the loss of NADPH-nitrate reductase activity and to the constitutive synthesis of nitrite reductase in the absence of the inducers, nitrate or nitrite. A minority of cnx and niaD mutants retain an inducible nitrite reductase activity (Pateman et al., 1964). MacDonald
NITRATE ASSIMILATION IN EUKARYOTIC CELLS
15
et al. (1974) examined the subunit structure of nitrate reductase in wild-type, niaD and c m mutants by studying the sedimentation coefficients of the NADPH- cytochrome c reductase (NADPH-CR) activity of nitrate reductase. They found that the wild-type NADPH-CR had a sedimentation coefficient of 7.6 S (200,000 MW), whereas certain mutant strains had NADPH-CR of 4.5 S (90,000-100,000 MW). To explain these results, they proposed that the niaD gene specifies an NADPH-CR of 4.5 S and the nitrate reductase molecule is a dimeric molecule of two 4.5 S niaD products. cnx mutants which constitutively synthesized nitrite reductase were shown to have a NADPH-CR with a 4.5 S sedimentation coefficient, whereas most c m mutants with an inducible nitrite reductase had both the 7.6 S and 4.5 S species of NADPH-CR. The only exceptions to these findings were mutants at the cnxE locus. Mutants of this locus had either inducible or constitutive nitrite reductase activity, but only the 7.6 S species of NADPH-CR. Since cmE mutants are the only mutants in which exogenous molybdate will partially restore NADPH-nitrate reductase (Arst et al., 1970), it seems likely that cmE mutants are capable of synthesizing the metal-free cofactor which is then able to cause dimerization of two 4.5 S subunits to form 7.6 S enzyme. To explain the findings that cllx and niaD mutants have either inducible or constitutive synthesis of NADPH-CR and nitrite reductase, it was proposed that the nitrate reductase molecule somehow regulated its own synthesis. Such an autoregulatory model has been presented by Cove and Pateman (1969). Mutations which affect the integrity of the nitrate reductase enzyme such as c m or niaD mutants produce nitrate reductase constitutively. Mutants that do not affect the structural integrity of the nitrate reductase show wild-type patterns of inducibility for NADPH-CR and nitrite reductase (Section II1,B). The crn nitrate transport mutant ofA. nidulans is discussed in Section IV,A,8.
2. Neurospora crassa Recently, Tomsett and Garrett (1980, 1981) have undertaken an extensive genetic study of nitrate assimilation in N . crassa, complementing the biochemical study of Coddington (1976). Both studies indicate that the genetics and biochemistry of nitrate assimilation are very similar between N. crassa and A . nidulans (Table 111). The nit-3 gene encodes the nitrate reductase apoprotein and nit-6 apparently encodes the nitrite reductase apoprotein. The nit- 1, nit-7, nit-8, and nit-9 genes lack both nitrate reductase and xanthine dehydrogenase activity and appear very similar to cnx mutants of A. nidulans. nil-9 mutants fall into three complementation groups in a manner identical to that found in cnxABC mutants of A. nidulans. The biochemistry of molybdenum cofactor loci is less well known in N. crassa, and no molybdate-repairable locus similar to that of cmE or regulatory locus such as cnxJ has yet been identified. It is not known if one of the molybdenum cofactor loci in N . crassa shares a structural role similar to cmH, as tentatively proposed for A. niduluns. The nit-3 and nit-6 loci encod-
16
NIGEL S. DUNN-COLEMAN ET AL
ing nitrate reductase and nitrite reductase in N . crussu are on different linkage groups, unlike the situation in A. nidulans where niaD and niiA are very tightly linked. The nitrate assimilation enzymes in both these fungi have been shown to be inducible, and the increase in activity observed when inducer (nitrate or nitrite) is present is almost certainly the result of de n o w protein synthesis. Bahns and Garrett (1980) transferred uninduced cultures of N . crassa to medium containing 90% deuterium oxide plus nitrate and found that the nitrate reductase formed was of a uniformly heavy density. In the reciprocal experiment where mycelia were grown under noninducing conditions on media prepared in 80% D,O and then induced with nitrate in H,O-based media, nitrate reductase displayed a normal density. This result indicates that there can be little, if any, inactive or precursor nitrate reductase protein in cultures not exposed to nitrate or nitrite. Amy and Garrett (1979) used monospecific antibodies against N . crassa nitrate reductase and showed that uninduced wild-type cultures had no detectable cross-reacting material, again arguing against any inactive or precursor nitrate reductase being present under uninduced conditions. It is obvious from these results that nitrate reductase is under strict regulatory control. Several genes have been identified which affect the expression of nitrate assimilation enzymes (Table IV). The nirA and nit-4/5 loci of A . nidulans and N . crassa, respectively, play a role in the induction of the nitrate assimilation pathway (see also Section III,B), whereas the areA and nit-2 loci of A. nidulans and N . crassa, respectively, are concerned with nitrogen metabolite repression of a variety of pathways including nitrate assimilation (discussed in Section 111,C). Recessive mutations in the nirA or nit-4/5 genes result in noninducibility of nitrate reductase and nitrite reductase. A second class of semidominant mutant (nirA") at the nirA gene results in constitutive expression of these enzymes in the absence of inducers. (nirA" mutants are still subject to nitrogen metabolite repression of nitrate reductase and nitrite reductase.) nirA" mutants have a higher constitutive expression of nitrate reductase compared with nitrite reductase. This is an important finding because the niaD and niiA loci are very tightly linked in A. nidulans and there is the possibility that they may be coordinately regulated in the manner of an operon such as seen in prokaryotes. The differential expression of nitrate reductase and nitrite reductase in nirA" mutants limits this possibility. In addition, Arst et al. (1979) described a nonreciprocal translocation mutant, nis-5, which has a portion of linkage group I1 translocated into the region between the niaD and niiA genes (linkage group VIII). This mutant synthesizes 40% of the wild-type level of nitrite reductase upon induction but also constitutively synthesizes 8% of induced enzyme levels. Arst et al. explained these results by suggesting that the normal promoter for nitrite reductase is between the niaD and niiA genes and the translocation in nis-5 inserted a second promoter responsible for the low constitutive expression of nitrite reductase.
TABLE IV FUNGAL MUTANTSWHICHAFFECTTHE EXPRESSION OF NITRATE REDUCTASE A N D NITRITE REDUCTASE ~~~~
~~~
N . crussa
A. niduluns
Function
nit-415
nirA
Positive regulator required for the induction of nitrate reductase and nitrite reductase
nirA-/nit-415 nirAC nirAd
nit-2
areA
nmr- 1
Positive regulator required for expression of nitrate reductase and nitrite reductase. Action of this regulator mitigated by reduced nitrogen Two- to threefold elevation of nitrate reductase and nitrite reductase activities
urn
gdhA
NADP-GDH structural gene
gln- 1
glnA
ma-1
meaA, meaB
Probable glutamine synthetase structural gene Methylamine resistant, possibly altered in ammonium uptake and/or compartmentation
~
Comments
ureAr areAd
Noninducible mutants Constitutive expression of the enzymes, mutants of this class still subject to nitrogen metabolite repression Constitutive expression of the enzymes. Also insensitive to nitrogen metabolite repression. Selected as a suppressor of an areA'; nirACmutant Noninducible mutants Insensitive to nitrogen metabolite repression but require induction for expression
Four allelic mutants isolated with higher maximal levels of these enzymes and decreased sensitivity to nitrogen metabolite repression. Possibly altered for translational or hierarchical control of nitrate reductase and nitrite reductase synthesis Mutants at this locus grow poorly on ammonium and are not subject to ammonium repression of nitrate reductase and nitrite reductase Mutants may be insensitive to nitrogen metabolite repression Mutants at these loci are insensitive to ammonium repression of nitrate reductase and nitrite reductase
18
NlGEL S. DUNN-COLEMAN ET AL
3. Yeast At present, no nitrate reductase mutants have been reported in any yeast. The most widely studied yeast, Sacckarornyces cerevisiae and SckizoSaccharornyces pornbe, lack the ability to assimilate nitrate. However, the regulation of nitrate assimilation has been studied in Torulopsis nitru-atophila(Rivas et al., 1983) and Candida utilis (Prabhakara et al., 1976). Actinomycin D, an inhibitor of DNAdependent RNA synthesis, was shown by Prabhakara et al. to prevent the induction of nitrate reductase in C . utilis, in accord with the supposition that nitrateinduced synthesis of the enzyme requires transcription. Prabhakara's results also indicated that nitrate is not required for the translation of nitrate reductase mRNA. Thus, these results contrast with those of Sorger and Davies (1973) who concluded on the basis of studies employing inhibitors of macromolecular synthesis that nitrate was essential for translation of nitrate reductase mRNA, but not for the transcription of the nitrate reductase gene. Rivas et al. (1973) also showed that the T. nitratopkila system was similar to that of A . nidulans and N . crassa in that NADPH was the electron donor for nitrate reductase and nitrite reductase, in contrast to most plants where NADH and ferredoxin are favored. Zauner and Dellweg (1983) have recently reported the purification of nitrate reductase from Hansenula anornula.
B. AUTOGENOUS REGULATION To explain the findings that mutants such as niaD or cnx, which alter the structural integrity of nitrate reductase, result in constitutive expression of nitrate reductase, Cove and Pateman (1969) proposed an autoregulatory model. The recessive nature of nirA mutants in nirA- :nirA heterozygous diploids led Cove and Pateman (1969) to suggest that the nirA gene product plays a positive role in regulating the expression of both nitrate reductase and nitrite reductase. The finding that nirA" mutants are semidominant in nirAC:nirA heterozygous diploids (Cove, 1969) indicated that the nirA gene product was produced in limited quantities. Autogenous regulation can be rationalized as follows. Although nitrate reductase is an inducible enzyme, small but detectable quantities of the enzyme are present in cells in the absence of nitratdnitrite. In the absence of nitratdnitrite, nitrate reductase binds to the nirA gene product, thereby preventing the positive action of this gene product on niuD and niiA gene transcription. In the presence of nitrate, the nitrate reductasemitrate complex is unable to interact with the nirA gene product. As a consequence, transcription of nitrate reductase and nitrite reductase is activated. Mutations in either niaD or cnx genes which alter the overall structural integrity of nitrate reductase prevent the nirA gene product from binding, resulting in constitutive synthesis of both nitrate reductase and nitrite reductase. Tomsett and Garrett (198 1) found that certain N . crussa nit-3 mutants as well as molybdenum cofactor mutants such as nit-8 also ~
+
+
-
NITRATE ASSIMILATION IN EUKARYOTIC CELLS nit-3
&
nit-^
t
NaR apoprotein
19
nit-6
I
+
NiR apoprotein
I
y
Positive regulator J
Inactive complex
Nitrate reductase
+4
Nitrate reductase: NO; complex
FIG. I . Model for autogenous regulation of nitrate reductase in N . crussa. The nif-4/5 gene produces a positive regulatory element necessary for nit-3 and nit-6 expression. The nit-3 gene produces a protein which in conjunction with a functional molybdenum cofactor produces the native nitrate reductase molecule. If nitrate is absent, the nitrate reductase binds and sequesters the nit-415 product. Mutations in nit-3 or the molybdenum cofactor genes (nit 1,7,8,9) can result in an aberrant nitrate reductase incapable of interaction with the nif-4/5 regulator. Then, the nit-3 and nit-6 genes appear constitutively expressed.
have constitutive expression of nitrite reductase and nitrate reductase activities. The model for autogenous regulation is depicted in Fig. 1. As Marzluf (1981) points out, the proposed autoregulatory role for nitrate reductase, although accounting for the biochemical and genetical data, must remain speculative in the absence of direct molecular biological evidence.
C. NITROGENMETABOLISM REPRESSION OF NITRATEASSIMILATION The ammonium generated from a variety of pathways, including nitrate assimilation, is used by cells to form glutamine. First, glutamate is generated from 2oxoglutarate and ammonium via the action of NADP-specific glutamate dehydrogenase (NADP-GDH). Another equivalent of ammonium is then consumed through the action of glutamine synthetase (GS) converting glutamate to glutamine. IN. crassa (Hummelt and Mora, 1980; Dunn-Coleman et al., 1981b) and A . nidulans (Garrett and MacDonald, unpublished observations) also possess low levels of glutamate synthase (GOGAT) which forms glutamate from glutamine, 2-oxoglutarate and NADH. GOGAT is apparently important in these fungi only when NADP-GDH is absent or ammonium is limiting.] In addition to regulatory control over the expression of nitrate assimilation under conditions of induction, a second control mechanism limits the expression of this pathway and other ammonium-producing pathways when cells are grown with metabolites such as ammonium or glutamine. This phenomenon is called nitrogen metabalite repression and has been extensively studied in both A . nidulans and N . crassa. The nit-2 locus of N . crassa and the equivalent areA locus of A . nidulans play
20
NlGEL S. DUNN-COLEMAN ET AL.
key regulatory roles in the mediation of nitrogen metabolite repression in these fungi (Table IV). Both the nit-2 and areA genes are believed to encode positive trans-acting regulatory products required for the expression of genes which are subject to nitrogen metabolite repression. Pateman and Kinghorn (1977) had proposed that the tamA locus of A . nidulans played a role equally important as the areA locus in nitrogen metabolite repression. However, Arst et al. (1982b) have recently shown that tumA plays no major role. Two types of mutants have been isolated at the areA locus. The most common class, designated areA' (r for repressed), leads to an inability to utilize nitrogen sources other than ammonium or glutamine. A much rarer class, designated areAd (d for derepressed), results in specific derepressed expression of certain of the enzymes, such as nitrate reductase, which are usually subject to nitrogen metabolite repression. No derepressed mutants at the nit-2 locus have yet been isolated. This apparent lack may be the result of greater technical difficulties in isolating mutants in N . crassa. The isolation of several types of auxotrophic mutants impaired in the utilization of nitrogen has provided insights ino the mechanism of nitrogen metabolite repression and possible modes of action of the areA and nit-2 loci (see Table IV). Mutants possibly impaired in the uptake of ammonium are resistant to its toxic analog, methylamine; examples are meaA and meaB mutants in A. nidulans (Arst and Cove, 1969) and mea-1 in N . crassa (Dunn-Coleman and Garrett, 1984). These mutants have derepressed levels of enzymes such as nitrate reductase when grown with ammonium. Further, structural gene mutants for NADPGDH in both A . nidulans (gdhA) and N . crassa (am) are also insensitive to ammonium repression. These results led to the proposal by Pateman et al. (1973) that the NADP-GDH protein itself plays a regulatory role in ammonium repression. However, Dantzig et al. (1978) showed that am mutants, although insensitive to ammonium repression of nitrate reductase, have repressed levels of this enzyme when grown with amino acids such as glutamate. From these and other findings, it was evident that ammonium had to be assimilated before repression could occur. Dantzig et al. proposed the term nitrogen metabolite repression to accommodate these results. Glutamine synthetase in N . crassa has been extensively studied by Mora and Palacio's group (Davila et al., 1978, 1980, and references therein). They found that the oligomeric structure of this enzyme was dependent on the nitrogen status of the cell. When N . crassa was grown under conditions of nitrogen limitation, a tetrameric form of glutamine synthetase was present in the cell. Cells grown under conditions of nitrogen excess had an octameric form of the enzyme. Recently these researchers have shown that two similar but distinct monomers, a and p, may be involved in the formation of the different oligomers of glutamine synthetase. N . crassa grown under ammonium limitation has a tetrameric form of the enzyme being composed of a-subunits, whereas the octameric form of the GS found in cultures grown under nitrogen
NITRATE ASSIMILATION IN EUKARYOTIC CELLS
21
sufficiency consists of the P-subunit polypeptide (Davila et al., 1980). The subunit structure of glutamine synthetase in the two mutants gln- l a and gln-lb has been shown to be devoid of the P-polypeptide (Sanchez et ul., 1980). However, both of these mutants retain significant levels of glutamine synthetase activity, either as the tetrameric form in gln-la or both tetrameric and dimeric forms in the more extreme gln-lb mutant. Sorger’s group (Premakumar et al., 1979) and Garrett’s group (Dunn-Coleman et al., 1981a) have examined nitrogen metabolite repression in gln-la and gln-lb. Both gln-la and gln-lb have derepressed levels of nitrate reductase and nitrite reductase when grown in the presence of ammonium or glutamate. However, in gln- la, glutamine represses these enzymes. In contrast, gln- l b had varying levels of derepressed expression of these two enzymes when grown with glutamine. These findings with gln-lb (Dunn-Coleman et al., 1981a) stand in contradiction to those of Premakumar et al. (1979) who found that gln-lb had repressed levels of nitrate reductase when grown with glutamine. Two models have been suggested to explain nitrogen metabolite repression in N . crassa and A. nidulans. In the first, it has been proposed that glutamine itself is the effector of nitrogen metabolite repression by directly preventing the positive action of the nit-2 (or areA) gene product. One can envision a concentration-dependent interaction of glutamine and the nit-2 gene product, which is hypothesized to be a glutamine-binding protein. Presumably, once the concentration of glutamine reaches a sufficient value, its interaction with the nit-2 product would lead to a complex no longer capable of causing an activation of the genes encoding enzymes of pathways under nit-2 control. Evidence to support this view comes from Grove and Marzluf (1981) who reported the isolation of a nonhistone chromosomal protein from N . crussu nuclei having properties consistent with such a role for the nit-2 gene product. Grove and Marzluf radioactively labeled isolated nonhistone chromosomal proteins from N . crassa nuclei and chromatographed them on a N . crassa DNA-cellulose affinity column. After washing the column to remove nonspecifically bound material, they employed glutamine to elute any glutamine-binding proteins and found a single glutaminebinding, DNA-binding protein (MW 22,000 polypeptide). Two nit-2 mutant strains were shown to have diminished amounts of this protein. Marzluf (1981) postulated that glutamine, by directly interacting with the nit-2 gene product, reduced its binding efficiency for DNA enough that transcription of structural genes under nit-2 control (such as nitrate reductase) would not be activated. A second model to explain nitrogen metabolite repression has been put forward by Dunn-Coleman et al. (1979, 1980, 1981a). This model implicates a role for glutamine synthetase itself in mediating nitrogen metabolite repression. The principal justification for this model resides in the observation that the mutant gln-lb has substantially derepressed levels of both nitrate reductase and nitrite reductase when grown with glutamine. If glutamine were the direct effector of
22
NIGEL S. DUNN-COLEMAN ET AL.
nitrogen metabolite repression, one would expect gin-1 b to behave like wild-type and gln- l a and have repressed levels of nitrate assimilation enzymes when grown with glutamine. Dunn-Coleman et al. (1979, 1980, 1981a) proposed that the octameric form of glutamine synthetase which is found under conditions of nitrogen sufficiency directly or indirectly inactivates the nit-2 product. Nitrogen metabolite repression is thus understood as follows. When nitrogen becomes limiting, the tetrameric form of glutamine synthetase supercedes the octameric form in the cell, thereby freeing the nit-2 gene product to activate nitrate reductase gene expression. The summary diagram for the regulation of nitrate assimilation in N . CYUSSU presented in Fig. 2 encompasses these various interpretations. One of the difficulties in interpreting the role of glutamine and glutamine synthetase in nitrogen metabolite repression through gln mutant studies is that both gln-la and gln-lb have substantial amounts of glutamine synthetase activity. Recently, MacDonald (1982) has isolated a glutamine synthetase mutant
+
-iH4
NO~-NO;
I ,~[r
g
2-oxoglutarate
y
NADP-GOH
nit-1
&-7
*-8
G-9ABC
nit-4/5
fi-6
I’
&-2; 8
w
w Trans-acting positive regulatory gene for NO;/ NO2induction
G-31
I I/
tet--rttt-cF--c-c
Trans-acting positive regulatory gene, inactivated by nitrogen sufficiency (nitrogen metabolite repressed)
FIG. 2 . Model for the regulation of nitrate assimilation in N . crussu. Expression of the structural genes, nir-3 and nir-6 which encode the nitrate assimilation enzymes, nitrate reductase (NaR) and nitrite reductase (NiR) respectively, requires the action of two regulatory genes nit-415 and nit-2. nit-4/5 acts when nitrate (or nitrite) is present to serve as inducer; nit-2 acts only when nitrogen metabolite repression is relieved, i.e., the reduced nitrogen reserves are low. Glutamine and perhaps glutamine synthetase (GS) are essential for nitrogen metabolite repression. n i f - I , 7, 8, and 9 function in molybdenum acquisition and molybdenum cofactor (MoCo) biosynthesis. Nitrate reductase is dependent on MoCo for its physiological activity.
NITRATE ASSIMILATION IN EUKARYOTIC CELLS
23
in A . nidulans, &A-I, which lacks all glutamine synthetase activity. MacDonald found that this mutant had derepressed levels of nitrate reductase when grown with nitrate and glutamine. MacDonald’s results also tend to indicate that glutamine itself is not simply the corepressor of the areA or nit-2 gene product and would implicate glutamine synthetase as having a role in mediating nitrogen metabolite repression. However, as Marzluf (1981) points out, glutamine could be sequestered in a pool or compartment where it plays a role in nitrogen metabolite repression and this pool may be inaccessible to exogenously supplied glutamine. A genetic way to resolve the role of glutamine synthetase in nitrogen metabolite repression is as MacDonald (1982) suggested; that is, to map mutations which both abolish glutamine synthetase activity and relieve nitrogen metabolite repression, and see if they lie within the glutamine synthetase structural gene. Very recently, Gonzalez et al. (1983) studied the intracellular distribution of glutamine. Only 12% of the glutamine present in wild-type cells was sequestered in vesicles. In contrast, 50-60% of cellular arginine is in vesicles. These results tend to indicate that most glutamine is available for nitrogen metabolite repression. D. POSSIBLETRANSLATIONAL CONTROLOF NITRATEASSIMILATION Recently, another locus was identified in N . crassa which affects the expression of both nitrate reductase and nitrite reductase. Mutations at this locus, termed nmr-1 , were characterized by their sensitivity to the toxic analog chlorate even when glutamine is the nitrogen source (Table IV). [Under these growth conditions, wild-type cells have repressed levels of nitrate reductase and are resistant to chlorate (Dunn-Coleman et al., 1981a; Tomsett et al., 1981).] These mutants have up to threefold higher levels of nitrate reductase and nitrite reductase compared with wild-type. Immunoelectrophoretic determinations showed that the higher nitrate reductase activities in nmr-1 mutants were due to greater enzyme concentrations. Also, the half-life of nitrate reductase in these mutants was unaltered. Reduced nitrogen metabolites such as ammonium, glutamate, and glutamine retained the capacity to repress nitrate reductase in these mutants (Table V). Similar repression of nitrite reductase also occurs in nmr-1 mutants. Double mutants constructed between nmr-1 mutants and the regulatory gene mutants nit-415 and nit-2 indicated that these genes were epistatic to nmr-I. A reasonable interpretation is that the nit-2 and nit-4/5 genes exert their positive effects at the level of transcription of the nitrate reductase and nitrite reductase structural genes (nit-3 and nit-6, respectively). One possible explanation is that the nmr-1 gene product also acts on nit-3 and nit-6 expression at the transcriptional level. Several considerations militate against this view. First, all four known mutations in nmr-1 lead to enhanced enzyme levels, the inference being that loss of function in nmr-1 is the likely consequence of mutation and that such
24
NIGEL S. DUNN-COLEMAN ET AL. TABLE V THERESPONSE OF NITRATE REDUCTASE I N WILD-TYPE A N D nmr-l MUTANTS TO VARIOUS NITROGEN SOURCES" NaN03
Wild type nmr-1 (114) nmr-1 (183) nmr-1 (304) nmr-1 (319)
100 181 145 245 154
NaN03
+ NH4CI 40 45 49 66 38
NaN03
+ [.-glutamate 55 46 69 43 41
NaN03
+ L-glutamine 5 3 6 13 3
OThe results are expressed as the percentage wild-type nitrate reductase specific activity under conditions of maximum induction, 37.0 nmol nitrite producedlminlmg protein. Results adapted from Dunn-Coleman et al. (1981a).
loss in function does not reconcile with enhanced transcriptional activity. Second, a transcriptional role for nmr- 1 would implicate a third positive control element in the genetic expression of nitrate assimilation which would not be attended by any underlying physiological basis for its action (as in nitrate induction for nit-4/5, or nitrogen metabolite repression for nit-2). The possibility that a hierarchical arrangement of regulation exists between nmr-1 , nit-2, and or nit-4/5 merit consideration. Although the original studies on nmr- 1 afford no insight in this regard, the possibility that nmr-1 encodes a repressor antagonistic to either nit-2 or nit-4/5 would be compatible with this observation. The epistasy of nmr-1 to nit-3 is not only evidenced by enhanced nitrate reductase expression with the wild-type nit-3 allele but also with a mutant nit-3 allele (no. 14789) which has enhanced levels of the partial nitrate reductase activity, MVH-nitrate reductase. Another possible explanation for nmr-1 gene function is that its gene product acts at a posttranscriptional level on nitrate assimilation. Possibly, enhanced rates of translation of nitrate assimilation enzyme-specific mRNAs or increased rounds of translation of these messages would account for the results. Premakumar et ul. (1978) have shown that N . crussu nitrate reductase mRNA has a half-life of only 8.5 minutes. The higher nitrate reductase and nitrite reductase in nmr- I mutants may result because their specific mRNAs are more stable in these mutants and greater amounts of enzyme can be synthesized. That is, the nmr-1 gene product may govern nitrate assimilation mRNA degradation. This possibility has not been tested and the precise role of nmr-1 remains unknown. Nevertheless, the specificity of its effects in enhancing the enzymes if nitrate assimilation is striking. Using an experimental strategy suggested by the studies of Tomsett et al. (1981), Sorger's laboratory (Premakumar et al., 1980) subsequently isolated an unusual N . crussu mutant also altered in nitrate reductase regulation. This single mutant, MS5, had elevated levels of nitrate reductase, nitrite reductase, histidase, and acetamidase even in the presence of glutamine.
NITRATE ASSIMILATION IN EUKARYOTIC CELLS
25
(The latter two enzymes were not examined in nmr-1 mutants by Dunn-Coleman et al., 1981a.) Sorger’s results also showed that the half-life of nitrate reductase in MS5 was significantly altered. The MS5 mutant has now been mapped was shown to be an allele of nmr-1 (Debrusk, unpublished).
E. EVIDENCE FOR
THE
TURNOVER OF NITRATE REDUCTASE
Cove (1966) showed that the absence of inducer and the presence of ammonium caused rapid loss of nitrate reductase activity in wild-type cells of A . nidulans. Hynes (1973) and Dunn-Coleman and Pateman (1977) confirmed and extended these findings, showing that cycloheximide, a protein synthesis inhibitor, did not significantly reduce this loss of nitrate reductase activity. Amy and Garrett (1980) obtained similar results in N . crassa and showed that when mycelia were exposed to ammonium plus cycloheximide, nitrate plus cycloheximide or nitrogen-free media or media lacking a carbon source, the amount of material cross reactive with anti-nitrate reductase antibodies declined in concert with nitrate reductase activity. Amy and Garrett concluded that the loss of nitrate reductase activity was attributable to the loss of this protein. It seems likely that proteolytic degradation of nitrate reductase occurs. Walls et al. (1978) and Sorger et al. (1978) have attempted to examine the nature of this proteolysis in N . crassa. They presented evidence for two different decay mechanisms, a general protein turnover system (designated N) and a second system (designated A) which increased in proteolytic activity with mycelial age. Sorger et al. also presented in vitro evidence for two different nitrate reductase inactivator systems, I and 11. Inactivator I was present in all mycelia and may correspond to system A. Inactivator I1 was nitrogen repressible and missing in nit-2 mutants and may correspond to system N. Lewis and Fincham (1970) studied the regulation of nitrate reductase in the basidiomycete Ustilago maydis. Ammonium ions were shown to repress nitrate reductase activity. Cycloheximide and actinomycin D (an inhibitor of mRNA synthesis) led to maintenance of nitrate reductase activity even in the presence of ammonium. Lewis and Fincham further showed that the loss of nitrate reductase activity in U . maydis upon exposure to ammonium was attributable to turnover of this protein, not a reversible enzyme inactivation. F. THEEFFECTSOF CARBON METABOLISM ON NITRATEASSIMILATION Both nitrate reductase and nitrite reductase in fungi use NADPH as electron donor. Hankinson and Cove (1974) examined the regulation of the pentose phosphate pathway (PPP) in A . nidulans, which generates NADPH via the action of glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH). Growth of wild-type A . nidulans in media containing urea plus nitrate resulted in a twofold elevation of G6PDH and 6PGDH activities
26
NIGEL S. DUNN-COLEMAN ET AL
compared with levels in mycelia grown with urea alone. Hankinson and Cove (1974) also examined various nitrate assimilation mutants and showed that the mutant noninducible for nitrate assimilation, nirA , did not show any elevation of PPP activities. On the other hand, niaD nitrate reductase apoprotein mutants and cnx molybdenum cofactor mutants could be divided into two classes. One class of niaD and c m mutants had elevated pentose phosphate pathway enzymes when grown with nitrate. The second class did not show such enzyme elevation. Hankinson and Cove (1974) proposed that the nirA+ product is responsible for the increases in the activities of the pentose phosphate pathway enzymes. Hankinson (1974) also described two mutants @ppA andpppB) which were impaired in their ability to reduce NADP+. These two mutants utilized nitrate poorly. Dunn-Coleman and Pateman ( 1979) examined hexokinase and phosphoglucomutase (PGM) activities in A . nidulans. Hexokinase activities did not vary with changes in carbon or nitrogen source. However, PGM activities were low in wild-type cells grown with nitrate and higher when cells were grown on ammonium. These findings seemed surprising at first, for it was anticipated that nitrate-grown cells would have elevated hexokinase activity to supply increased amounts of glucose 6-phosphate to the pentose phosphate pathway for NADPH generation. The findings were rationalized by a consideration by PGM regulation. PGM activity is lower in nitrate-grown cells, thus the rate of conversion of glucose 6-phosphate to glucose 1 -phosphate is lowered, allowing greater utilization of glucose 6-phosphate by the pentose phosphate pathway and a consequent enhancement of NADPH production. Schwytzer and Garrett (unpublished) examined pentose phosphate pathway activity regulation in N . crassa and were unable to find elevated enzyme levels in mycelia grown with nitrate. They concluded that the flux of glucose through the pentose phosphate pathway could vary with the nitrogen source such that cellular NADPH demands were met without changes in enzyme activities. ~
IV. Genetics of Nitrate Assimilation in Plants A. GENETICANALYSIS Two approaches have been successfully used to isolate nitrate reductase mutants in different species of plants (Tables VI and VII). The first has been the use of plant tissue culture, in which cell suspensions or protoplasts are screened for resistance to chlorate, the toxic analog of nitrate (Alberg, 1947). The second approach has been to mutagenize seed (M1 generation) and screen seedlings in the segregating M2 generation for low nitrate reductase activity. This approach, in the case of cereals such as barley (Hordeum vulgare), overcomes one of the serious difficulties with cereal tissue culture, that is, the lack of success in
NITRATE ASSIMILATION IN EUKARYOTIC CELLS
27
regenerating plants from cell suspension or protoplasts. Both approaches have yielded nitrate reductase structural gene mutants and molybdenum cofactor mutants, but no loci analogous to the regulatory loci seen in fungi have yet been described in higher plants. 1. Nicotiana tobacum and Nicotiana plumbaginijolia Nitrate Reductuse Mutants Miiller’s group at Gatersleben (GDR) was the first to successfully use tissue culture to select for nitrate reductase mutants in allodihaploid ( n = 24) cell cultures of Nicotiana tobacum (Muller and Grafe, 1978; Mendel and Muller, 1979, 1980). Their approach was to either mutagenize with ENU or select for spontaneous resistance to chlorate in wild-type cell suspensions. To date, they have isolated 40 variant cell lines, 36 of which are defective in the nitrate reductase apoprotein (niaA) and 4 lines defective in the synthesis of the molybdenum cofactor (cnxA). From the work with A . nidulans and N . c r a m , it is known that at least 9 genes are involved in nitrate assimilation. To overcome a possible problem of isolating a wide range of recessive mutants in N . tobacum, Maliga’s group in Szeged (Hungary) have recently used N. plumbaginifolia as a model system to isolate nitrate reductase mutants. Mutant isolation was carried out using haploid protoplast cultures, and 4 different types of nitrate reductase mutants ( 1 apoprotein mutant and 3 cofactor mutants) were isolated (Marton et al., 1982). It is interesting to note that Muller and Grafe (1978) treated 2 X lo7 cells with 0.25 mM ENU and isolated 5 nitrate reductase-defective lines. Maliga’s group used 8 X lo4 protoplast-derived colonies with 0.3 mM ENU and found 92 chlorate-resistant colonies (Marton et al., 1982). Only a small fraction of the chlorate-resistant clones in N . plumbaginijolia completely lack nitrate reductase, whereas most of the N . tabacum niaA mutants have no detectable enzyme activity. Miiller and Mendel (1982) have now regenerated fertile mutant plants from 15 niaA lines. They found that the nitrate reductase deficiency in these mutants is the result of two unlinked recessive mutations. Thus they concluded that NADH-nitrate reductase in N . tobacum is encoded by duplicate structural genes which they named niaAl and niaA2. The presence of duplicate genes for nitrate reductase in N. tobacum is probably the result of its allodihaploid nature (Miiller, 1983). Six chlorate-resistant lines (NA-type) of N. plumbaginijolia have similar characteristics to niaA mutants of N . tobacum. They are strictly auxotrophic on nitrate media and have xanthine dehydrogenase activity. Two of the mutants (NA8 and NA14) retain a residual amount of nitrate reductase activity. Unlike the niaA mutants of N . tobacum, the N . plumbaginijolia mutants are probably the result of a single mutation. Mendel and Miiller (1979) have also studied the biochemistry of nitrate assimilation in wild-type and mutant cells of N . tobacum. The electron donor for nitrate reductase is NADH; NADPH will not function. There does not appear to
28
NIGEL S. DUNN-COLEMAN ET AL
be either a biphasic NAD(P)H-nitrate reductase or a NADPH-nitrate reductase present in N. tobacum. None of the niaA mutants had any detectable NADH-, FADH,-, or BVH-nitrate reductase activity, with the exception of niaA-95 which retained some FADH,- and BVH-nitrate reductase activity. Cytochrome c reductase (NADH-CR) activities were low in all niaA mutants and this activity was not inducible by nitrate. Mendel and Miiller (1979, 1982) have shown that nitrate reductase and nitrite reductase activity are inducible in wild-type cells, confirming the original observation of Filner (1966). Cycloheximide prevented induction indicating that the increase in activity was likely due to de novo protein synthesis. Nitrite reductase levels in the niaA mutants were similar to those of the wild-type, indicating that nitrate reduction is not needed for the induction of nitrite reductase. Miiller and Mendel (1982) also examined nitrate reductase activity in the niaA1/2 double mutant seedlings. Ten niaA1/2 mutant lines were incapable of growth with nitrate as the sole nitrogen source and showed a nitrogen-starved appearance. These results indicate that NADH-nitrate reductase is essential for the utilization of nitrate as the sole nitrogen source. Two other niaA1/2 mutants (129 and 134) retained a residual nitrate reductase activity. Seedlings of these mutants grew on nitrate at reduced rates, initially having 5% and eventually 60% of the wild-type in vivo nitrate reductase activity (determined by nitrite accumulation). Yet, Miiller and Mendel observed that cultured cells derived from these mutants behaved like strict auxotrophs on nitrate medium. One possible explanation which may account for the nitrate reductase activity observed is in vivo complementation between defective nitrate reductase polypeptides produced by the mutated niaA1 and niaA2 genes. Cultured cells may not have sufficient time to produce enough nitrate reductase to grow on nitrate, whereas seedlings may slowly accumulate nitrate reductase and become capable of utilizing nitrate. More recently, Evola (1983) has isolated a chlorate-resistant cell line (clr 19) which lacks nitrate reductase activity and thus may be allelic to the niaA loci. Two other variants, clr 15 and clr 30, had lower resistance to chlorate and could still assimilate nitrate. 2 . Molybdenum Cofactor Mutants in Nicotiana and Datura Two types of molybdenum cofactor mutants have now been isolated in N . tobacum, the cnxA mutant isolated by Muller’s group and a second cnx type recently isolated by Buchanan and Wray (1982) (Table VII). Mutants in either cnxA or the second cnx locus (called cnxB in Table VII) lack both nitrate reductase and xanthine dehydrogenase. However, cnxA mutants are molybdate repairable both in vivo and in vitro; that is, when grown with high concentrations of molybdate, they develop 30% of the fully induced wild-type level of nitrate reductase (xanthine dehydrogenase activity is also partially restored). Grafe and Miiller (1983) have recently shown that the four cnxA mutants are alleles of the
NITRATE ASSIMILATION IN EUKARYOTIC CELLS
29
same pair of duplicate loci (cnxA1 and cnxA2). The cnxB-type mutants are not molybdate repairable. The molybdenum cofactor from cmA, but not cnxB, mutants is capable of restoring NADPH-nitrate reductase activity in cell-free extracts of the N . crassa nit-l molybdenum cofactor mutant. The cnxA mutant also differed from the wild-type with respect to the regulation of the enzymes in nitrate assimilation. The inactive nitrate reductase and the nitrite reductase were both found to be constitutively expressed (Muller and Mendel, 1982). These observations are similar to those found in the fungi A . nidulans and N . crassa (Cove, 1979; Tomsett and Garrett, 1981). This result indicates that the tobacco nitrate reductase is autogenously regulated as in fungi (see Section 111,B). Further, it regulates expression of nitrite reductase as well. This conclusion derives from the crucial observation that mutants which affect the structural integrity of the enzyme alter the pattern of regulation of those two enzymes. The molybdenum cofactor mutants of N . plumbaginifolia are less well characterized. The NX 1 mutant is molybdate repairable and will complement the N . crassa nit- 1 mutant in vitro; it therefore appears similar to the cnxA mutant of N . tobacum. The two other molybdenum cofactor mutants of N . plumbaginifolia, NX 21 and NX 24, are not molybdate repairable and are not allelic since somatic hybrids derived from fusing protoplasts have restored nitrate reductase activity (Marton et al., 1982). NX 24 is equivalent to cnxB of N . tobacum (Xuan et al., 1983). Mendel et al. (1982~)studied the regulation of molybdenum cofactor synthesis in N . tobacum. Molybdenum cofactor content was determined by the ability of a cell-free extract of tobacco to restore NADPH-nitrate reductase activity to an extract of N . crassa nit- 1. The addition of nitrate to an amino acidgrown culture of wild-type cells resulted in a concurrent increase in both nitrate reductase and molybdenum cofactor content of the cells. This result was unlike that found by Garrett and Cove (1976) in A . nidulans, where nitrate did not influence molybdenum cofactor content. But as Mendel et al. (1982~)point out, Garrett and Cove measured “free” cofactor, while Mendel et al. assayed total molybdenum cofactor content following heat treatment of cofactor-containing extracts. Four cnxA mutants were examined and all were shown to have a constitutively maintained high molybdenum cofactor content. The niaAl/2 mutants could be divided into two groups depending on molybdenum cofactor content. Group 1 niaA1/2 mutants had a low molybdenum cofactor content, and the levels were not inducible by nitrate. Group 2 niaA1/2 mutants had molybdenum cofactor levels similar to wild-type. Recently, Evola ( 1 983) has isolated a chlorate-resistant variant (clr0) in N . tobacum lacking both nitrate reductase and xanthine dehydrogenase activity. This variant may be allelic to either cnxA or CUB. A possible molybdenum cofactor mutant has been isolated in Datura innoxia by King and Khanna (1980). The variant cell line, N R l , was selected from a haploid cell culture grown in the presence of chlorate. The NR1 mutant was
30
NlGEL S . DUNN-COLEMAN E T AL
unable to grow on nitrate as the sole nitrogen source and lacked NADH-nitrate reductase activity. The mutant did however retain NADH-cytochrome c reductase activity typical of that seen in cnxA mutants of N . tobacum. Recently, molybdenum cofactor mutants also have been isolated in Hyoscyamus muticus by King’s group ( S t r a w et al., 1981; Gebhardt et al., 1982). A total of five clones lacking nitrate reductase activity were isolated from MNN9-mutagenized haploid protoplasts. Unlike previously described methods for isolating nitrate reductase mutants by resistance to chlorate, King’s group let the protoplasts from colonies on enriched media and then plate-tested a sample from each colony on selective media. This procedure also lead to the identification of various amino acidrequiring clones, e.g., tryptophan. All four clones lacked nitrate reductase activity. Xanthine dehydrogenase activity was examined and shown to be absent in three clones: VIC2, I,D12, and XIVE9. The clone I,D12 was also shown to have molybdate-repairable nitrate reductase and xanthine dehydrogenase and is therefore similar to cnxA of N . tobacum. Gebhardt et al. (1982) tentatively concluded that the four clones belonged to at least two different complementation groups. 3. Barley and Pea Nitrate Assimilation Mutants Kleinhofs and Warner’s group (Washington State University) have isolated nitrate reductase structural gene and molybdenum cofactor mutants in both barley (Hordeum vulgare) and pea (Pisum sativum) (Tables VI and VII). The strategy used to isolate the mutants in both plants was similar. Mutations were induced by incubating seeds with the mutagen NaN, (M1 generation). The treated seed was then planted, selfed, and the segregating M2 generation seedlings individually assayed in vivo for nitrate reductase activity (Warner er al., 1977, 1982; Kleinhofs et al., 1978; Warner and Kleinhofs, 1981). Three nitrate reductase-deficient mutants were isolated in pea. Two of the mutants which were shown to be allelic, A317 and A334, retained less than 6%of the wild-type level of nitrate reductase and had normal levels of xanthine dehydrogenase activity. These mutants appear to be nitrate reductase apoprotein mutants and the designation nar-1 was given to them. The third mutant (A300) lacked xanthine dehydrogenase activity and retained only 20% of the wild-type level of nitrate reductase. This mutant was designated as nar-2 and appears to be a molybdenum cofactor mutant. Warner et al. (1982) undertook a genetic analysis of the mutants and showed that nar-1 and nar-2 are nonallelic. F, hybrids between each mutant and the wild-type (Juneau) had intermediate levels of nitrate reductase activity, indicating incomplete dominance of the deficiency. The F, segregation of the mutants indicated that the nitrate reductase deficiency was controlled by a single nuclear gene. Feenstra and Jacobsen (1980) and Feenstra et al. (1982) have also isolated a nitrate reductase mutant in pea. They used an isolation procedure similar to that of Warner and Kleinhofs and also showed the mutant to be chlorate resistant. As in the case of the nar-1 mutants, Feenstra’s mutant (El)
TABLE VI NITRATEREDUCTASE STRUCTURAL GENEMUTANTSI N PLANTS
Plant cell tissue culture
Locus or mutant number
Method of isolation
Comments
References
N . tobacum cell cultures are allodihaploid and Miiller’s group has recently shown that niaA mutants are in fact unlinked double mutants niaAl, niaA2. niaA mutants are strictly auxotrophic on nitrate as the sole nitrogen source NA (equivalent to niaA mutants of N . tobacum) were isolated in haploid protoplast cultures and are likely to be single mutants. NA mutants are strict auxotrophs on nitrate as the sole nitrogen source nar-1 mutants retain 3-10% of wild-type NADH-nitrate reductase activity. These mutants grows on nitrate due to a second, bispecific NAD(P)H-nitrate reductase enzyme Two allelic mutants, A317 and A334, retain less than 6% wild-type level of nitrate reductase activity
Miiller and Mendel (1982); Miiller (1983)
The mutant 305 may be allele of nitA
Nichols and Syrett (1978); Sosa et al. (1978) Braaksma (1982)
Nicotiana tobacum
nid
ENUO mutagenesis or spontaneous resistance to KCIO, in cell suspensions
Nicotiana plumbaginifolia
NA
Either spontaneous, 6OCo irradiation, NEUU mutagenesis in protoplasts, and screening for resistance to KCIO,
Hordeum vulgare
nar-l
NaN, mutagenesis (Ml) of seeds screening M2 progeny for low nitrate reductase activity
Pisum sativum
nar-1
Chlamydomonas reinhardii
nitA
NaN, mutagenesis (Ml) of seeds screening M2 progeny for low nitrate reductase activity UV irradiation, screening for KCIO3 resistance
Arabidopsis thaliana
chl-2 chl-3
aENU and NEU, N-ethyl-N-nitrosourea
EMS mutagenesis (Ml) M2 progenies were screened for KC103
The mutants B21 (chl-2 locus) and B29 (chl-3) locus) retain 30 and lo%, respectively, of the fully induced wild-type level of nitrate reductase
Marton et al. (1982)
Dailey et al. (1982a,b); Warner et al. (1977); Kleinhofs et al. (1983); Kuo et al. (1984) Warner et a / . (1982)
TABLE VII MOLYBDENUM COPACTOR MUTANTSIN PLANTS
Plant species Nicotiana tabacum
w
Nicotiana plumbaginifolia
Hyoscyamus muticus
Datura innoxia
Locus or mutant number
Xanthine Molyb- In vitro dehydate compleNitrate drogen- KC103 repair mention reductase ase ac- resistant/ of de- of nit-1 Method of isolation activity tivity sensitive fect mutant
EMUa mutagenesis of cell suspension and then selection for chiorate resistance EMU mutagenesis CnxB of cell suspension and then selection for chlorate resistance NXl (cnxA) Spontaneous, 6OCo irradiation or NX21 (cnxC) NEU" mutaNX24 (cnxB) genesis of protoplasts then selection for KC103 resistance MNNG mutagenesis I,D 12 of protoplasts, VIC2 then screening for XIVE9 nitrate nonutilizaMA-2 tion Spontaneous reNR 1 sistance to KC103 in cell suspension cnxA
0
0
R
+
+
0
0
R
-
+
0 0 0
0 0 0
R R R
+
+
-
? ?
0 0 0 0
0 0
R R R R
0
7
0 0
R
-
+ -
+ ?
? ? 0
Comments
Reference
Four allelic mutants, Muller and Grafe constituitive (1978); Mendel and NADH-CR, and Muller ( 1979); derepressed MoCo Muller and Mendel synthesis ( 1982) Four allelic mutants, Buchanan and Wray inducible NADH( 1982) CR and MoCo syn- Mendel et al. (1984) thesis NXl similar to cmA. Marton et al. (1982) NXI, NX21, and Mendel et al. (1982a) NX24 are nonallelic mutations, somatic hybrids prototrophic 12D12 and MA-2 are similar to cnxA and NXI. At least two different loci Lacks FMNH2-NR, MVH-NR activity. Has NADH-CR activity
Gebhardt er al. (1 982) Strauss et al. (1981) Lazar et al. (1983) Fankhauser et al. ( 1984) King and Khanna ( 1980)
Physcomirrella paiens
CitX
Arabidopsis thaliana
Cn*
rgn Pisum sativum
Hordeum vulgare
nar-2
Cn*
W
nar-2
nar-3
Chlamydomonas reinhardii
nit-C
NTG mutagenesis of spores and then selection for KC103 resistance NTG mutagenesis (Ml) of seeds, then screening M2-segregating population for NaR activity NaN3 mutagenesis (Ml), screening M2 progeny for NaR activity NaN3 mutagenesis (MI), screening M2 progeny for NaR activity NaN3 mutagenesis (Ml), screening M2 progeny for NaR activity
EMS mutagenesis screening M2 progeny for chlorate resistance UV irradiation
aNEU and EMU, N-ethyl-N-nitrosourea.
0
0
R
1 of 3
?
Three cornplementation groups
Ashton and Cove (1977); Dunlop et al. (1982)
10%
10-
R
+
?
1%
15% 10%
R
-
?
Braaksma and Feenstra (1982)
20%
0
?
?
?
Warner et al. (1982)
1%
0
R
?
?
Bright ef al. (1983)
13%
0
S
?
?
10%
0
R
?
?
0
0
R
?
?
Formerly A ~ 3 4
Warner and Kleinhofs (1981); Kuo et al. (1983; Dailey et al. (1982); Kleinhofs el al. (1983); Tokarev and Shumny (1977) Formerly Xno 18, not Narayanan et al. allelic to nar-2 (1984)
Lacks MVH-NR activity, has NADHCR
Nichols and Syrett (1978)
34
NIGEL S. DUNN-COLEMAN ET AL
retained only 5% of the wild-type level of nitrate reductase and was inherited as a monogenic trait. Feenstra et al. (1982) also studied the effect of nitrate on symbiotic nitrogen fixation in both wild-type (c.v. Rhondo) and E l . Evidently, supply of nitrate to leguminous plants actively fixing nitrogen decreases the rate of N, fixation. Nitrogen fixation, as judged by acetylene reduction, was only inhibited 19% in the mutant E l compared with 47% in the wild-type when grown in the presence of nitrate, indicating that nitrate has to be reduced by the plant before its inhibitory effect on nitrogen fixation can take place. The nitrate reductase mutants isolated in barley by Kleinhofs and Warner’s group appear very similar to those they isolated in pea. Two unlinked loci have been identified. The nar-1 locus appears to be the structural gene for the nitrate reductase apoprotein, and the nar-2 locus, a molybdenum cofactor gene. Genetic analysis of both loci indicates that they are each inherited as a single nuclear gene. Mutants at nar-1 locus are codominant and nar-2 mutants are recessive. Tokarev and Shumny (1977) have also isolated three barley nitrate reductase mutants by EMS mutagenesis and chlorate resistance screening. The mutant Xno 29 is an allele of nar- 1. The other two mutants, Xno 18 and Xno 19, are allelic to one another but are not allelic to either nar- I or nar-2. Kleinhofs et al. proposed that a new locus, nar-3, is defined by the Xno 18 and Xno 19 mutants. These nar-3 mutants lack xanthine dehydrogenase activity and therefore appear to be molybdenum cofactor mutants (Somers et ul., 1983). Narayanan et al. (1983) have recently examined the nature of cytochrome c reductase (CR) in wild-type and nitrate reductase mutants of barley. They found that nur mutants with significant CR activity fall into two classes, one had a 8 S enzyme species, the other had only the 4 S CR form. Barley nitrate reductase is apparently a homodimeric protein with 100 kd subunits. Although the barley nar-1 mutants retain less than 10% of both the in vivo and in vitro nitrate reductase activity of the control, they are capable of substantial nitrate reduction and produce as much dry weight and protein content as control plants. One explanation for these results is that there is a bispecific NAD(P)H-nitrate reductase activity in barley. Dailey et al. (1982b) have looked for such an activity in nar-la mutants. Using Affi-Gel blue affinity chromatography which does not bind wild-type NADH-nitrate reductase, they purified a bispecific NAD(P)H-nitrate reductase from the mutant nar- la. This bispecific nitrate reductase probably accounts for the residual nitrate reductase activity seen in nar-1 mutants, and consequently, their ability to utilize nitrate as a nitrogen source. Kleinhofs et al. (1983) have attempted to select for mutants lacking the NAD(P)H-nitrate reductase. They mutagenized seed from the nar- l a mutant and screened for M2 seedlings lacking nitrate reductase activity. Three mutants were isolated which grew poorly on nitrate and were deficient in xanthine dehydrogenase activity also. They are likely to be molybdenum cofactor mutants. A similar mutant has been isolated by Bright et al. (1983), using NaN, mutagenesis of barley (cv. Mario Mink). This mutant (R9401) is the result of a single recessive mutation and has 1% of the wild-type in vitro nitrate
NITRATE ASSIMILATION IN EUKARYOTIC CELLS
35
reductase activity. R9401 is chlorate resistant, unlike the mutants isolated by Warner and Kleinhofs, and lacks xanthine dehydrogenase activity. It is unable to grow on nitrate as sole nitrogen source either in soil or sterile culture and appears to be a conditional lethal c m type mutant. At present, it is not known if this mutant or the mutants isolated by Kleinhofs et al. define a new locus or a more extreme allele of the nar-2 locus. In both barley and pea, the nar-1 and nar-2 mutants had nitrate reductase specific activities inducible by nitrate at higher than maximal levels in wild-type. Notton et al. (1983) have recently shown that the mutant nar-la contains both the nitrate reductase apoprotein and molybdenum cofactor. The nitrate reductase deficiency in these mutants appears to be due to an inability to maintain nitrate reductase in an active aggregated form. Similar results have been reported by Asan et al. (1983). 4. Arabidopsis thaliana
Arabidopsis thulianu has also been used as a model system to study nitrate assimilation. The advantages of this plant include a short generation time, haploid chromosome number of 5 , self-fertility , and homozygous “wild-type” material (Braaksma, 1982). Chlorate-resistant mutants were generated by treating seeds with either the mutagen EMS or NTG (MI generation). The M1 generation plants were selfed and M2 seedlings grown in the presence of chlorate. Plants which showed resistance or little damage from chlorate were characterized further (Braaksma and Feenstra, 1979, 1982; Braaksma, 1982). A total of 61 chlorate-resistant mutants were isolated, 10 of which had lowered nitrate reductase activity. The remaining 51 mutants were allelic (chl-1 locus) and were impaired in the uptake of chlorate. The mutants which were impaired in their nitrate reductase activity were divided into 7 different complementation groups. Two mutants, B25 and B73, also had low xanthine dehydrogenase activity, and the lesion in B73 was partially repaired by exogenous molybdate. The mutant B73 therefore appears similar to cmA of N . tobacum, NX 1 of N . plumbaginifolia, and cnxE of A . nidulans. A second mutant B25 (rgn locus), which had very low levels of both the NADH-nitrate reductase and FMNH,-nitrate reductase activity, also appears to be defective in molybdenum cofactor biosynthesis. None of the nitrate reductase mutants had constitutive expression of nitrite reductase. Two other chlorate-resistant complementation groups, chl-2 and chl-3, had normal levels of xanthine dehydrogenase activity but lowered levels of nitrate reductase activity. Either or both of these loci could encode the nitrate reductase apoprotein. The functions of the other chlorate-resistant mutants are unknown. 5. Soybean Recently, soybean mutants lacking constitutive nitrate reductase activity have been isolated (Nelson et al., 1983; Ryan et al., 1983). Approximately 12,000 seedlings isolated from mutagenized seed were screened for chlorate resistance
36
NIGEL S. DUNN-COLEMAN ET AL.
and low nitrate reductase activity. Three mutants were isolated (NR-2, NR-3, and NR-4) and shown to be allelic. Nitrate reductase activities were determined in seedlings grown with either nitrate or urea as nitrogen source. When the mutants were grown on nitrate, they had approximately 50% of the activity found in nitrate-grown wild-type seedlings. However, urea-grown mutants had no detectable nitrate reductase activity. Nelson et al. (1983) concluded that the nitrate reductase activity observed in leaves of nitrate-grown soybean plants consists of both inducible and constitutively expressed activities. They also observed that the mutants lacked an associated NO,,, evolution normally found in young leaves of wild-type plants.
6. Physcomitrella patens Ashton and Cove (1977) isolated three chlorate-resistant mutants in the moss Physcomitrella patens, designated nat- 1, nat-2, and nat-3. All three mutants grew poorly when either nitrate or nitrite was the sole nitrogen source. Unlike nat-3, the nat-1 and nat-2 mutants grew like the wild-type on uric acid or hypoxanthine as a nitrogen source. However, as Ashton and Cove pointed out, P. patens utilizes purines very poorly. More recently, Cove’s group has isolated 34 more nitrate nonutilizing mutants in P. patens (Dunlop et al., 1982; Dunlop, 1982) of which 27 have been partially characterized. Eight of these show leaky growth while 19 do not grow at all on nitrate. Of these 19, 12 have been characterized as putative cnx-type mutants since they lack both nitrate reductase and xanthine dehydrogenase activity. Further, all 12 have constitutive expression of nitrite reductase activity. Somatic hybrids produced by protoplast fusion have so far identified at least three complementation groups among this dozen cmtype mutants. One appears molybdate repairable. No nitrate reductase structural gene mutants have been isolated and Dunlop et al. (1982) speculated that the nitrate reductase gene may be duplicated in P. patens. Their finding that the cnxtype mutants demonstrate constitutive expression of nitrite reductase is consistent with the findings of Muller and Mendel (1982) in N . tobacum, Tomsett and Garrett (1981) in N . crassa, and Cove (reviewed in 1979) in A. nidulans. That is, mutations which affect the structural integrity of the nitrate reductase enzyme, such as cm or apoprotein mutants, may produce nitrate reductase and nitrite reductase constitutively (see Section II,B above). 7. Algae Nitrate assimilation mutants have also been isolated in green algae. Huskey et al. (1979) have identified three genes, nit-A, nit-B, and nit-C, in Volvox carteri which are affected in nitrate assimilation. The nit-A and nit-C mutants lack both NADH-nitrate reductase and NADH-cytochrome c reductase activities and may be defective in the formation of nitrate reductase (either the apoprotein or molybdenum cofactor) or in the regulation of nitrate reductase. The nit-B3 mutant,
37
NITRATE ASSIMILATION IN EUKARYOTIC CELLS
unlike nit-A or nit-C types, is not resistant to chlorate and retains some nitrate reductase activity. Levels of nitrate reductase activity in nit-B3 are a function of the nitrate concentration in the media, suggesting that the nit-B gene may play a role in nitrate uptake. Nichols and Syrett (1978) and Nichols et al. (1978) UV irridated Chlamydomonas rienhardii cells and selected for chlorate resistance. Three loci were identified, mutations in which resulted in failure to utilize nitrate as the sole nitrogen source. One, nit-C, when mutated was also unable to use hypoxanthine as a nitrogen source and thus appears to be a molybdenum cofactor mutant. The nit-A gene mutant lacked both NADH-nitrate reductase and NADH-cytochrome c reductase activities but retained BVH-nitrate reductase activity. It is probably a mutant in the nitrate reductase apoprotein structural gene. The nit-B mutant had reduced transport of nitrate. Nichols et al. (1978) concluded that nit-B could be a regulatory locus for nitrate assimilation. Similar mutants to those reported by Nichols and Syrett (1978) have been described by Sosa et al. (1978) and biochemically characterized by Fernandez and Cardenas TABLE VIII POSSIBLECANDIDATES FOR NITRATE UPTAKEMUTANTS Organism
Mutant/locus/cell line
Characteristics
Aspergillus nidulans
crnA
Volvox carteri
nitB
Nicotiana tobacum
0 41
Arabidopsis thaliana
chl- I
KCIO3 resistant. Has nitrate reductase and nitrite reductase activity KCIO, resistant. Level of nitrate reductase activity is related to the external nitrate concentration KCIO, resistant. Initially unable to grow normally on nitrate, eventually both nitrate reductase and nitrite reductase levels higher than the w i Id-type KC103 resistant. Reduced uptake of chlorate. Normal levels of nitrate reductase and nitrite reductdse
Reference Tomsett and Cove (1979); Brownlee and Arst (1983) Huskey et a / . (1979)
Qureshi et a / . (1982)
Braaksma (1982)
38
NlGEL S. DUNN-COLEMAN ET AL
(1981a,b). In addition, Sosa et al. (1978) described a fourth class of mutants having normal levels of nitrate reductase enzyme activities but unable to grow on nitrate. These could possibly be nitrate transport mutants (see below).
8. Transport Mutants Several possible candidates for nitrate transport mutants have been isolated in plants and fungi (see Table VIII). They are all typified by their resistance to chlorate and retention of nitrate reductase activity. The chlorate-resistant cell line (041) of N . tobacum (Quershi et al., 1982) exhibited a lag of 15 days in cell culture before exponential growth on nitrate was achieved. Maximal levels of both nitrate reductase and nitrite reductase were found after 34 days, whereas wild-type cells exhibited expotential growth and maximal enzyme levels after only 4 days. The results in N . tobacum and also in Volvox carteri (Huskey et al., 1979) indicate that diffusion of nitrate is sufficient to enable survival of nitrate transport mutant cells. Recently, Brownlee and Arst ( 1983) have biochemically characterized nitrate uptake in A. niduluns. The crnAl mutation reduced nitrate uptake dramatically in conidiophores and young mycelia but did not influence uptake in older mycelia. The crnA locus is part of the niaD niiA nitrate assimilation gene cluster and is controlled by the areA gene product but not the nirA gene product. Differential control suggests that the crnA niiA niaD gene cluster is not expressed as a tricistronic messenger. B . CONCLUSIONS Two important classes of mutants have yet to be isolated or well characterized in plants. First, no nitrite reductase structural mutants have been isolated. Such mutants have been isolated only in fungi, based upon their failure to grow on either nitrate or nitrite as a nitrogen source. These mutants are not chlorate resistant (because they still have nitrate reductase activity) but can be recognized by replica plating from a reduced nitrogen source such as ammonium onto either nitrate or nitrite. Replica plating is not possible yet in plant tissue culture and this may explain why such mutants have not been isolated. One approach to isolating nitrite reductase mutants in plants would be to screen plants for high in vivo accumulation of nitrite, but high concentrations of nitrite may be very toxic and such plants may die at an early stage of growth. An alternative approach deserving consideration is the protocol of King et nl. (1982). They used a mutant screening procedure with haploid mesophyll protoplasts from H . muticus. Protoplasts surviving mutagenesis were allowed to proliferate in complete medium and then were transferred as clones to minimal medium. Clones which failed to grow on minimal medium were characterized further. A total of 14 clones with specific stable defects were identified.
NITRATE ASSIMILATION IN EUKARYOTIC CELLS
39
Although regulatory mutants affecting the enzymes of nitrate assimilation have been found in several fungi (e.g., the nit-2 and nit-415 loci in N . crassa), no such mutants have been isolated in higher plants. The work of Filner (1966), Muller and Mendel (1982), and others has shown that the nitrate assimilation pathway is both inducible by nitrate and repressible by reduced nitrogen. Also, protein synthesis inhibitors such as cycloheximide prevent an increase in nitrate reductase activity. These results indicate that the nitrate assimilation pathway is under strict regulation, probably at the level of transcription. Tomsett and Garrett (1980) isolated 67 nitrate assimilation mutants in N . crassa, either on the basis of chlorate resistance or failure to grow on nitrate as the sole nitrogen source. Six mutants were isolated at either the nit-2 or nit-415 regulatory loci. Miiller and Mendel (1982) have, to date, isolated 40 nitrate reductase mutants in N . tobacum. However, the mutants are of only two types: niaA apoprotein mutants (36 mutants) or cnxA (4 mutants). No putative regulatory mutants were isolated. These results may reflect problems in isolating recessive mutants in amphihaploid cell culture. Maliga’s group (Marton et al., 1982) have identified four different kinds of mutants (1 nia and 3 cnx-type mutants) in haploid N . plumbaginifolia protoplasts in 9 characterized clones. The use of haploid material as a source material appears to increase the probability of identifying a greater range of mutants. So far, the molybdenum cofactor mutants and apoprotein mutants appear to be analogous to similar mutants found in fungi. It can, therefore, only be a matter of time before the existence of regulatory mutants affecting nitrate assimilation in higher plants is determined.
V. Cell and Molecular Biological Advances in Nitrate Assimilation A. SOMATICHYBRIDIZATION STUDIESIN NITRATEASSIMILATION Nason et al. (1970) initially demonstrated the in vitro complementation of nitrate nonutilizing mutants by the restoration of NADPH-nitrate reductase activity upon cohomogenization of mycelia from a nit- 1 (molybdenum cofactor) mutant and a nit-3 (apoprotein) mutant of N . crassa (Section 11,F). This in vitro complementation was then achieved with higher plants by Mendel and Miiller (1979) who in vitro reconstituted NADH-nitrate reductase activity of N . tobacum using mutant cell lines of the niaA (apoprotein) and cnxA (molybdenum cofactor) mutants. These successes led to in vivo complementation by somatic hybridization in higher plants. Glimelius et al. (1978) selected for somatic hybrids using PEG-mediated fusion of protoplasts of cnxA and niaA mutants of N . tobacum. The hybrid cells had restored nitrate reductase activity, the result of genetic complementation, and were able to utilize nitrate as the sole nitrogen source. However, plating niaAlcnxA fused protoplasts directly onto nitrate was
40
NIGEL S. DUNN-COLEMAN ET AL
unsuccessful in isolating somatic hybrids. The fused protoplasts had to be first plated onto nonselective medium (containing amino acids) for several days until cell walls had regenerated and small clusters of cells had developed. These clusters of cells (mini-calli) were then plated onto nitrate-containing media and the proliferating calli were purified. Shoots could be regenerated from these hybrid cell lines, unlike the parental cell lines which were unable to regenerate shoots. Marton et al. (1982) isolated somatic hybrids between nine nitrate reductase mutants of N . plumbaginifolia. The five apoprotein-defective (NA) cell lines were found to be noncomplementing and are therefore probably allelic. Somatic hybrids were obtained from the fusion of NA and NX (molybdenum cofactor) mutants and they showed restoration of nitrate reductase activity. These results were therefore identical to those of Glimelius et al. (1978) for N . tobacum. Marton et al. (1982) also found that the four NX cell lines belonged to three complementation groups (NXl and NX9, NX21, NX24). Somatic hybrids which had restored nitrate reductase activity also had regained the ability to regenerate shoots. Dunlop (1982) has also used somatic hybridization to isolate diploid hybrids for complementation studies in the moss P hyscomitrella patens and has identified at least three complementation groups among their putative cnx mutants (see Section IV,A,6). Lazar et al. (1983) have used protoplast fusion to isolated intergeneric somatic hybrids between a Hyoscyamus muticus nitrate reductase variant (MA-2) and the niaA( 115) and cnxA(68) mutants of N . tobacum. Only the MA-2/niaA( 115) combination yielded prototrophic intergeneric hybrids capable of growth on nitrate. Lazar et al. (1983) therefore concluded that the MA-2 variant had a defective molybdenum cofactor. When N . tobacum is grown in the absence of nitrate, nitrate reductase activities are only 20-30% of the fully induced, nitrategrown level. H . muticus however has undetectabie nitrate reductase activities when grown in the absence of nitrate. The intergeneric hybrids Lazar et al. (1983) isolated displayed the H . muticus pattern of nitrate reductase activity regulation. Gupta et al. (1983) have reported the correction of the cnxA(68) defect in protoplast derived-intergeneric gene transfers between the cnxA(68) mutant and either Datura innoxia or Physalis minima. Wild-type niesophyll protoplasts of D . innoxia and P . minima were X-ray irradiated, resulting in their becoming mitotically inactive and consequently unable to grow on nitrate. These protoplasts were then used with cnx(68) protoplasts and initially grown under nonselective conditions for several weeks and then plated onto nitrate media. A total of 45 nitrate-utilizing colonies were isolated from the nitrate medium. Since cnxA(68) is a nonrevertable mutant, Gupta et al. (1983) concluded that these colonies must have arisen from heteroplasmic fusions, and the frequency of successful transformation of the cmA(68) mutant was approximately 3%. The nitrate reductase activities of the cnxA(68): P . minima or D. innoxia transformants were 3-5% of the wild-type level in P. minima or D. innoxia. Gupta et al.
NITRATE ASSIMILATION IN EUKARYOTIC CELLS
41
(1983) suggested that inefficient expression of the D. innoxia or P . minima genes in the cnxA(68) cell line might account for the low levels of nitrate reductase activity. The results of Gupta et al. (1983) and Lazar et al. (1983) indicate that intergeneric complementation of nitrate reductase mutants is possible. It is a relatively straightforward technique to identify the hybrids of transformants by selecting for growth on nitrate after allowing such cells to undergo several rounds of cell division on non-selective medium. This step probably allows the hybrid or transformed cells to reach a sufficient population density so that there are enough viable cells to maintain growth when plated on selective media. Pental et al. (1982) used the N . tobacum mutant niaA( 130) to demonstrate the feasibility of identifying rare protophic clones among a background of nitrate nonutilizing niaA( 130) mutants. In the reconstruction experiments they conducted, a few wild-type protoplast-derived colonies were mixed with 4-5 X lo4 niaA( 130) colonies. Depending on the plant hormone composition of the medium used, nearly a 100% throughput of wild-type colonies could be identified. This work showed that it may be possible to select for plant transformation via functional complementation of mutant protoplasts using plant nitrate reductase mutants such as niaA(130). One can envisage using wild-type genomic cosmid libraries introduced into niaA( 130) protoplasts, via liposomes for example, and selecting for transformants capable of utilizing nitrate. This would provide one method of cloning plant nitrate assimilation genes. B. CLONING OF A MOLYBDENUM COFACTOR GENEFROM E . coli Taylor et al. (1982) have reported cloning the chlA molybdenum cofactor gene of E . coli. The presence of molybdenum cofactor in wild-type and chlorateresistant (chl)mutants of E . coli was determined by in vitro complementation of the nitrate reductase mutants of N . tobacum [cnxA(68)] and H . muticus (MA-2). Only the chlA mutant failed to complement either of the two plant nitrate reductase mutants. Taylor el al. (1982) and Kleinhofs et al. (1983) initially cloned chlA from a wild-type cosmid library which had been used to transform a chlA mutant. The cosmid clone (pJT1) was subcloned by HaeII deletion to a 10.8 kb plasmid designated pJ 13. Interestingly, this pJ13 clone would only transform the chlA mutant SA493 to wild phenotype; chlA mutant JP382 was not transformed by pJ13. By subcloning aHindIII fragment from pJTl into pBR322, Kleinhofs et al. were able to transform chlA SA493 to wild-type. Kleinhofset al. concluded that the chlA locus could be divided into two components. They proposed that the chlA locus should be changed to chlM (represented by the SA493 mutant) and chlN (represented by the JP382 mutant). Kleinhofs et al. also showed that the chlM gene encodes a polypeptide of 15,000 MW. Cell-free extracts of the chlA transformants were able to restore nitrate reductase activity to extracts of
42
NIGEL S. DUNN-COLEMAN ET AL
either N . tobacum cnxA(68) or H. muticus MA-2. With the development of a selectable plant transformation system for dicotyledonous plants (see Section V,D, below), it should be possible to insert the cloned chlA gene onto an appropriate vector to see if the chlA gene can complement mutants cnxA(68) and MA-2 in vivo. C. FUNGALTRANSFORMATION SYSTEMS
To date, three N . crassa genes and one A. nidulans gene have been cloned based upon their ability to complement equivalent E. coli mutants. In all cases, genomic libraries of these two fungi were constructed in the plasmid pBR322. Vapnek et al. (1977) reported the cloning of the qa-2 gene which encodes the catabolic quinate dehydrogenase (5-dehydroquinate hydro-lyase), due to its ability to complement an aroD E . coli mutant which lacked this activity. Schechtman and Yanofsky (1983) and Keesey and De Moss (1983) reported cloning the trp-1 gene of N . crassa via complementation of a trpC mutant of E. coli. Buxton and Radford (1983) were successful in cloning the orotidine 5'-phosphate carboxylase (pyr-4) gene of N . crassa by its complementation of the pyr F gene of E . coli. In A . nidulans, Kinghorn and Hawkins (1982) have cloned the synthetic dehydrogenase function of the arom cluster of A. nidulans via complementation of a aroD mutant of E. coli. All four of these cloned genes are apparently being expressed in E. coli due to fortuitous expression of an accompanying fungal promoter rather than expression via either of the two bacterial promoters found on the pBR322 plasmid. Only the qa-2 gene from the qa cluster of 4 genes is expressed in E . coli and it seems exceedingly likely that many genes of interest, particularly those of nitrate assimilation such as nitrate reductase and nitrite reductase, will not be able to be cloned via complementation of an equivalent E . coli mutant. A preliminary report citing evidence for the expression of the N . crassa nit-3 gene in E . coli (Smarrelli and Garrett, 1982) was erroneous and was traced to a Pseudomonas maltophilia contaminant. Case et al. (1979) reported the development of a cloning system for N . crassa based upon cotransformation of sequences upon complementation of a qa-2 mutant with the cloned qa-2 gene. Subsequent to this, Schweizer et al. (1981) reported using this transformation system to clone the remaining three genes of the 4a cluster (4a-1, 4a-3, and qa-4). N . crassa is believed to have no nuclear plasmids equivalent to the yeast 2 p DNA, which has been extensively used in yeast transformation experiments. The 2 p DNA plasmid is capable of autonomous replication and chimeric plasmids have been developed as yeast-E. coli shuttle vectors for cloning genes into either of the two. Selecting for stable N . crassa transformants using a genomic library in a plasmid which cannot replicate autonomously results in the selection of transformants with integrated vector DNA. This integration of the vector presents novel problems in reisolating the
NITRATE ASSIMILATION IN EUKARYOTIC CELLS
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gene of interest from the transformant. Recently, Stohl and Lambowitz (1983) reported the construction of a shuttle vector of N . crassa using one of the three mitochondria1plasmids of N . crassa. The report by Hughes et al. (1983) describing a chimeric plasmid which can replicate in E . coli and N . crassa was found to be erroneous. Development of a shuttle vector would greatly facilitate the cloning of N . crassa nuclear genes such as those concerned with nitrate assimilation. D. PLANTTRANSFORMATION Many species of dicotyledous plants are susceptible to crown gall disease, which is the result of infection of wound sites by the bacterium Agrobacterium tumefaciens. Recently, it has been shown that bacterial plasmids called Ti (tumor-inducing) are responsible for crown gall development and that a portion of the Ti plasmid, T-DNA, integrates into the plant nuclear genome. The genetic transformation of the plant cell by T-DNA results in the rapid proliferation of these transformed cells to form tumors (see Schell and Van Montagu, 1983, for a review). A consequence of this genetic transformation is the production by the plant cell of opines which are used by free-living A . tumefaciens. The genes coding for octopine and nopaline synthase are encoded in T-DNA. To overcome barriers to the expression of foreign genes in plants, a number of laboratories have constructed various chimeric genes consisting of promoter sequences derived from the nopaline synthase gene and various coding sequences such as the chloramphenicol acetyltransferase gene of pBR325, the niomycin phosphotransferase gene from Tn5, or the methothrexate resistance-conferring dihydrofolate reductase gene of the plasmid R67. These chimeric constructions have very recently been shown to be expressed in plant cells (Schell and Van Montagu, 1983; Bevan et al., 1983; Farley et al., 1983). With the development of a selectable system of plant transformation this should greatly facilitate the study of cloned nitrate assimilation genes reintroduced into plants. E. CLONING NITRATEASSIMILATION GENES-CONCLUDING
REMARKS
Several possible means exist to clone nitrate assimilation genes. First, the chlM molybdenum cofactor gene has already been cloned (Taylor et al., 1982), and it seems likely that additional E . coli genes for nitrate assimilation will be cloned shortly. In view of the demonstrated conserved nature of the molybdenum cofactor, it seems likely that with the appropriate regulatory sequences, molybdenum cofactor genes will be expressed in fungal and plant cells, thus providing a selectable means for plant transformation due to genetic complementation. With the development of a transformation system for N . crussa and the attendant use of shuttle vectors, it seems likely that many nitrate assimilation genes in N . crassa will be rapidly cloned. An extensive range of auxotrophic mutants for
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NIGEL S . DUNN-COLEMAN ET AL
nitrate assimilation genes is readily available. A similar case can be made for cloning A. nidulans nitrate assimilation genes. Tilburn et al. (1983) have reported the development of transformation of A. nidulans using the previously cloned Aspergillus acetamidase gene (see below), but transformation efficiencies were low. Hynes et al. (1983) have recently reported cloning the acetamidase structural gene (amdS) from A. nidulans. Using in vitro translation, Hynes et al. showed that amdS368 deletion mutant produced no amdS polypeptide, and it seemed likely that this mutant would produce no amdS mRNA. cDNA libraries were constructed from poly(A) mRNA isolated from the amdS368 deletion mutant and from a strain specially constructed to produce high levels of acetamidase. These libraries were used as probes to screen an A. nidulans library constructed in h phage (Charon 30). Three plaques showing strong hybridization to the cDNA from the enhanced amdS strain but not the amdS deletion mutant were isolated. Hynes et al. went on to map the amdS clones and showed that the controlling region of the gene is located at its 5’ end. Similar deletion mutants exist for the nitrate reductase and nitrite reductase genes (niaD and niiA) in A. nidulans. Tomsett and Cove undertook an elegant and extensive analysis of the niiA niaD gene region and deletion mapped closely linked niiA and niaD genes. An approach similar to that used to clone the amdS gene could be used to clone both nitrate reductase and nitrite reductase using these deletion mutants characterized by Tomsett and Cove (1 979). It is possible that cloned fungal nitrate assimilation genes may show enough DNA homology to plant genes to allow their use as probes for specific gene identification and isolation. A second approach would be to purify nitrate assimilation enzymes to homogeneity in quantities sufficient for microsequencing of the protein to be carried out. Knowledge of the amino acid sequence would allow the synthesis of oligonucleotide cDNA probes, assuming the protein sequenced does not have too many amino acids which have degenerate codon usage. Such cDNA probes could be used to screen genomic libraries of plants either in phage or cosmid constructions. This synthetic oligonucleotide approach has been successful in isolating the am gene (encoding NADP-GDH) from N . crassa (Kinnaird and Fincham, 1982). Alternatively, plant cosmid libraries could be shotgunned into plant protoplasts via liposomes with the hope that a gene of interest could be identified via complementation of an appropriate auxotrophic mutant. The development of transformation systems for Volvox and Chlamydomonas would allow the cloning of nitrate assimilation genes via complementation of existing mutants. Over the next few years, it seems exceedingly likely that the nitrate assimilation genes will be cloned and these achievements should provide great insights into the study of nitrate assimilation and its regulation.
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Quershi, J. A., Buchanan, R. J . , and Wray, J. L. (1982). Proc. Int. Symp. Nitrate Assimilat., Gatersleben (Abstr. ), Redinbaugh, M. G . , and Campbell, W. H. (1981). Plant Physiol. 68, 115-120. Redinbaugh, M. G . , and Campbell, W. H. (1983). Plant Physiol. 71, 205-207. Renosto, F., Omitz, D. M . , Peterson, D., and Segel, I. H. (1981). J . Biol. Chem. 256, 8616-8625. Rivas, J., Guerrero, M. G . , Paneque, A., and Losada, M. (1973). Plant Sci. Lett. 1, 105-113. Rossman, M. G . , Moras, D., and Olsen, K. W. (1974). Nature (London) 250, 194-199. Ryan, S. A . , Nelson, R. S . , and Harper, J. E. (1983). Plant Physiol. 72, 510-514. Rucklidge, G . , Notton, B. A., and Hewitt, E. J. (1976). Biochem. SOC. Trans. 4, 77-80. Sanchez, F., Calva, E., Compomanes, M . , Blanco, L., Guzman, J., Saboris, J. C., and Palacios, R. (1980). J. Biol. Chem. 255, 2231-2234. Schechtman, M., and Yanofsky, C. (1983). Neurosporu Newslett. 30, p. 19. Schell, J. and Van Montagu, M. (1983). Biorechnology 1, 175-180. Schrader, L. E., Ritenour, G. L., Eilrich, G. L., and Hageman, R. H. (1968). Plant Physiol. 43, 930-940. Schweizer, M., Case, M. E., Dykstra, C. L., Giles, N. H., and Kushner, S . R. (1981). Proc. Nutl. Acud. Sci. U.S.A. 78, 5086-5090. Scott, A. I., Irwin, A. J., Siegel, L. M . , and Schoolery, J. A. (1978). J. Am. Chem. Sac. 100,316318. Shen, T. C., Funkhouser, E. A,, and Guerrero, M. G . (1976). Plant Physiol. 58, 292-297. Sherrard, J. H., and Dalling, M. J. (1979). Plant Physiol. 63, 346-353. Sherrard, J. H., Kennedy, J. A,, and Dalling, M. J. (1979). Plant Physiol. 64, 640-645. Siegel, L. M., Davis, P. S . , and Kamin, H. (1974). J. Biol. Chem. 249, 1572-1586. Smarrelli, J . , Jr., and Campbell, W. H. (1981). Planr Physiol. 68, 1226-1230. Smarrelli, J., Jr., and Campbell, W. H. (1983). Biochim. Biophys. Acra 743, 435-445. Smarrelli, J., Jr., and Garrett, R. H. (1982). Fed. Proc. Fed. Am. Soc. Exp. Biol. 41, 756. Solomonson, L. P. (1974). Biochim. Biophys. Actu 334, 297-308. Solomonson, L. P. (1975). Biochim. Physiol. 56, 853-855. Solomonson, L. P., and Spehar, A. M. (1977). Nature (London) 265, 373-375. Solomonson, L. P., Lorimer, G . H., Hall, R. L., Borchers, R., and Bailey, J. L. (1975). J. Biol. Chem. 250,4120-4127. Somers, D. A., Kuo, T. M . , Kleinhofs, A., and Warner, R. L. (1983). Ptanrfhysiol. 71, 145-149. Sorger, G . J., and Davies, I. (1973). Biochem. J. 134, 673-685. Sorger, G. J., Premakumar, R., and Gooden, D. (1978). Biochim. Biophys. Actu 540, 33-47. Sosa, F. M., Ortega, T., and Barea, J . L. (1978). Plant Sci. Lett. 11, 51-58. Stewart, G. R., and Orebamjo, T. 0. (1979). New Phytol. 83, 311-319. Stohl, L. L., and Lambowitz, A. M. (1983). Proc. Narl. Acad. Sci. U.S.A. 80, 1058-1062. Strauss, A., Bucher, F., and King, P. J. (1981). Plunta 153, 75-80. Tachiki, T., and Nason, A. (1983). Biochim. Biophys. Acta 744, 16-22. Taylor, J. L., Kleinhofs, A., Bedbrook, J . R . , and Grant, F. I. (1982). Genetic Engineering: Applications to Agriculture. Abstract No. 41. Taylor, J. L., Bedbrook, J. R., Grant, F. J., and Kleinhofs, A . (1983). J . Molec. Appl. Genet. 2, 261 - 17I . Tilbum, J., Scazzocchio, C., Taylor, G., Lockington, A., Zabicki-Sissman, J. 0.. and Davis, R. W.(1983). Gene 26, 205-221. Tokarev, B. I . , and Shurnny, V. K. (1977). Genetika (Moscow) 13, 2097-2103. Tomsett, A. B., and Cove, D. J. (1979). Genet. Res. Camb. 34, 19-32. Tomsett, A. B., and Garrett, R. H. (1980). Genetics 95, 649-660. Tomsett, A. B., and Garrett, R. H. (1981). Mol. Gen. Genet. 184, 183-190.
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Tomsett, A. D., Dunn-Coleman, N. S., andGarrett, R . H. (1981). Mol. Gen. Genet. 182,229-233. Vapnek, D., Hautala, J . A., Jacobson, J . W., Giles, N. H., and Kushner, S. R. (1977). Proc. Natl. Acad. Sci. U.S.A. 74, 3508-3512. Vega, J. M. (1976). Arch. Microbiol. 109, 237-242. Vega, J. M., and Kamin, H. (1977). J . Biol. Chem. 252, 896-909. Vega, J . M., Garrett, R. H., and Siegel, L. M. (1975). J . Biol. Chem. 250, 7980-7989. Vennesland, B., and Guerrero, M. G. (1979). Encycl. Plant Physiol. New Ser. 6, 425-444. Wallace, W. (1974). Biochim. Biophys. Acta 341, 265. Wallace, W. (1975). Biochim. Biophys. Acta 377, 239-250. Walls, S., Sorger, G. J . , Gooden, D., and Klein, V. (1978). Biochim. Biophys. Acta 540, 23-32. Warner, R. L., and Kleinhofs, A. (1981). Plant Physiol. 67, 740-743. Warner, R. L., Lin, C. J., and Kleinhofs, A . (1977). Nurure (London) 269, 406-407. Warner, R. L., Kleinhofs, A , , and Muehlbaver, F. J. (1982). Crop Sci. 22, 389-392. Wilkerson, J. O., Janick, P. A , , and Siegel, L. M. (1983). Fed. Proc. Fed. Am. Soc. Exp. Biol. 42, 2060. Wrey, J. L., and Kirk, D. W. (1981). Plant Sci. Lett. 23, 207-213. Yamaya, T., Oaks, A., and Boesel, I. L. (1980). Plant Physiol. 65, 141-145. Xaun, L. T., Grafe, R . , and Muller, A. J. (1983). In?. Protoplast Symp. 6th, Basel, Switzerland (Abstr.). Zauner, E., and Dellweg, H. (1983). Eur. J . Microbiol. Biotech. 17, 90-95.
INTERNATIONAL REVIEW OF CYTOLOGY, VOL. 92
Endocytosis and Exocytosis: Current Concepts of Vesicle Traffic in Animal Cells MARKC. WILLINGHAM AND IRAPASTAN Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland I. Introduction ......... ........................... 11. Events at the Plasma Membrane.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . Receptors in the Plasma Membrane . . . . . . . . . . . . . . . . . . . . . . . B. Clustering in Coated Pits.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. The Structure of Coated Pits: Clathrin ..................... D. Vesicle Formation in Endocytosis from Coated Pits.. . . . . . . . . 111. Endocytic Vesicles: Characteristics. . A. Receptosomes ......................................... B. Multivesicular Bodies ............. D.
...........................
Tubular Elements
F. Macro- and Micropinocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Kinetic Classes of Endocytosis.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Compartmentalization in the Golgi . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Endocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . _.._.. B. Exocytosis: Materials Destined for the Cell Surface . . . . . . . . . . C. Lysosomal Delivery D. Materials from the Endoplasmic Reticulum.. . . . . . VI. Functions of Endocytic and Exocytic Membrane Traffic A. Recycling of Receptors and Ligands, Degradation . . . . . . . . . . . B. Traffic Control of Synthesized Materials . . . . . . . . . . . . . . . . . . . C. Pathologic Processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Hormonal Delivery?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Other Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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I. Introduction In recent years, a large amount of research in cell biology has concentrated on the events surrounding the binding, internalization, processing, and degradation of macromolecules that enter cells from their external environment (reviewed in Farquhar, 1983; Farquhar and Palade, 1981; Goldstein et al., 1979; Pastan and Willingham, 1981a,b, 1983; Pearse and Bretscher, 1981; Silverstein et al., 51 Copyright 0 1984 by Academic Press, Inc All rights of reproduction in any form reserved ISBN 0-12-364492-5
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1976; Steinman et al., 1983). This work has demonstrated that cells can selectively remove materials from their environment, and route these materials to different intracellular sites in a carefully controlled manner. Much of the data on the nature of the pathways taken by materials coming from the outside, as well as those originating from the inside, has been descriptive, but recently some of the molecular details of these processes have been uncovered. While there are still many unknown factors that control the shuttling of molecules between various membrane compartments, enough new information is available to justify a review of this subject and some speculation on possible mechanisms that might mediate these and related processes. The major thrust of many recent studies has been to understand how a cell can select from its surroundings a certain type of macromolecule, concentrate it on the cell surface, internalize it, and send it to a specific intracellular destination, where it may be degraded or returned intact to the cell surface. A considerable effort has also been devoted to the elucidation of how molecules synthesized within the cell are directed to the proper location. In some cases the same pathways appear to be involved. Much of this review will concentrate on work in our own laboratory as it relates to data in other systems. A schematic drawing summarizing the pathways to be discussed in this review is shown in Fig. 1.
P Me
Granule [Only in specialized
9 ‘ Cis
Nucleus
FIG. 1. Diagrammatic summary of the major pathways of endocytosis and exocytosis in animal cells. The receptors shown are examples of two of the different pathways possible: the solid receptor with its appropriate ligand (open square) represents the EGF receptor: the open receptor and its ligand (open triangle) represents the transferrin receptor.
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11. Events at the Plasma Membrane
A. RECEPTORS IN THE PLASMA MEMBRANE A wide variety of receptors are found on the surface of animal cells. Receptors have been identified for hormones and growth factors (Ackerman and Wolken, 1980, 1981; Ackerman et al., 1983; Ahmed et al., 1981; Bergeron et al., 1979; Carpenter and Cohen, 1976; Carpentier et al., 1978, 1979a,b, 1982; Cheng et al., 1980; Conn et al., 1978; Goldfine et al., 1977; Gorden et al., 1978a,b; Haigler et al., 1978, 1980a,b; Hillman and Schlessinger, 1982; Horiuchi et al., 1982, 1983; Kahn, 1976; King and Cuatrecasas, 1981; Levi et al., 1980; Maxfield et al., 1978, 1981a,b, 1979b; Orci e t a l . , 1978; Pastan rtal., 1981; Posner, et al., 1981; Varga et al., 1976), plasma proteins (Anderson et al., 1976, 1977, 1982; Ashwell and Morell, 1974; Bleil and Bretscher, 1982; Brown et al., 1982, 1983; Courtoy et al., 1982; Dautry-Varsat et al., 1983; Dickson et al., 1981a-c, 1982a-d, 1983; Fan et al., 1983; Geuze et al., 1983; Goldstein et al., 1979; Hanover et al., 1983, 1984; Iacopetta et al., 1983; Kaplan and Nielson, 1979; Kaplan et al., 1977; Klausner et al.. 1983; Maxfield et al., 1978, 1979a,b, 1981a,b; Neufeld and Ashwell, 1979; Neufeld et al., 1975; Pastan et al., 1977, 1981; Pastan and Willingham, 1981a,b; Pricer and Ashwell, 1971; Sando and Neufeld, 1977; Sly et al., 1981; Van Leuven et al., 1978; Via et al., 1982; Wall et al., 1980; Willingham et al., 1979, 1981a-c, 1983a-c; Willingham and Pastan, 1978, 1980, 1981a,b, 1982, 1983c,d), toxins (Donovan et al., 1981; FitzGerald et al., 1980, 1983a,b; Iglewski et al., 1977; Pappenheimer, 1977; Sandvig and Olsnes, 1980; Yamaizumi et al., 1978), viruses (Chardonnet and Dales, 1970; Dales, 1973, 1978; Dales and Choppin, 1962; Dales and Hanafusa, 1972; Dickson et al., 198Ic; Helenius et al., 1980; Helenius and Marsh, 1982; Marsh and Helenius, 1980; M a t h et al., 1982; Schlegel et al., 1982a,b, 1983; Simons et al., 1982; Simpson et al., 1969; Wehland et al., 1982a; Willingham et al., 1981a), and other substances found in the blood or other body fluids, as well as artificial materials (Abrahamson et al., 1979; Abrahamson and Rodewald, 1981; Adams et al., 1982; Bessis, 1963; Bessis and Breton-Gorius, 1962; Bretscher, 1983; Bretscher and Thomson, 1983; Bretscher et al., 1980; Fan et al., 1982; Farquhar, 1978, 1981, 1983; Fawcett, 1964; FitzGerald e t a l . , 1983b; Gonatas et al., 1977, 1980; Herzog and Farquhar, 1977; Herzog and Reggio, 1980; Joseph et al., 1978, 1979; Nagura et al., 1979; Nicolson, 1974; Ottosen et al., 1980; Petersen and van Deurs, 1983; Policard and Bessis; 1958; Rodewald, 1973; Rodewald and Abrahamson, 1980; Roth et al., 1976; Roth and Porter, 1962; Ryser et al., 1982; Salisbury et al., 1980; Stome, 1979; Willingham et al., 1979. 1981d; Woodward and Roth, 1978; Zoon et al., 1983). These receptors are generally, but not always, intrinsic membrane proteins that have a high affinity for a specific ligand.
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Much of our own work has concentrated on studying the binding and internalization of the plasma protein a,-macroglobulin (aZM) and its receptor. a2M is a tetrameric glycoprotein present in plasma which has the ability to complex tightly with a wide variety of proteases (Heimburger, 1979; van Leuven et al., 1978). On complexing with a protease or upon chemical reaction with amines, a,M becomes modified to a form that is recognized by a specific receptor on the surface of many mesenchymal cells, including many lines of tissue culture fibroblasts. These cultured cells have up to 5 X lo5 receptors per cell (Dickson et al., 1981b; Hanover et al., 1984). About 10-20,000 have a very high affinity; the rest have somewhat lower affinity. a,M is conveniently made from outdated human plasma. The human form of the protein binds well to the receptor on rodent cell lines (Pastan ef al., 1977). The large size of this protein (750,000 daltons) makes chemical derivitization of this molecule relatively easy. Another ligand that has been extensively studied is low-density lipoprotein (LDL) for which there are specific receptors on cultured fibroblasts (Anderson et al., 1977; Goldstein et al., 1979; Handley et al., 1981; Vermeer et al., 1980; Via et al., 1982). It was in the early morphologic studies with labeled LDL that selective localization of this ligand was seen in the coated pits of the plasma membrane of fibroblasts (Anderson et al., 1976, 1977). Accumulation in coated pits has been seen for many ligands bound to their receptors (Abrahamson and Rodewald, 1981; Ackerman et al., 1983; Bleil and Bretscher, 1982; Bretscher and Thomson, 1983; Brown et al., 1983; Carpentier et al., 1982; Courtoy et al., 1982; FitzGerald er al., 1983a,b; Iacopetta et al., 1983; McGookey et al., 1983; Pastan and Willingham, 1981a,b, 1983; Rodewald and Abrahamson, 1980; Roth et al., 1976; Salisbury et al., 1980; Via er al., 1982; Wall et al., 1980; Willingham et al., 1979, 1981a,c, 1983b; Willingham and Pastan, 1980, 1981a,b, 1982, 1983c,d; Zoon et al., 1983). For some ligands, the initial distribution of unoccupied receptors appears to be diffuse over the entire plasma membrane. The lateral mobility of some of these receptors when labeled with bound ligands has been measured by the fluorescence photobleaching recovery technique (Axelrod et al., 1976; Edidin et al., 1976; Hillman and Schlessinger, 1982; Jacobson and Poste, 1976; Maxfield et al., 1981a,b; Schlessinger et al., 1978). These studies show that most of these receptors move randomly and rapidly in the plane of the plasma membrane. These data indicate that at 37°C each receptor will move through one of the 1000 or so coated pits found on most types of cultured cells in about three seconds. Thus, the distribution of receptors can change rapidly. Evidence for the LDL system has suggested that many of the receptors for LDL are concentrated in coated pits in the absence of added ligand (Anderson et al., 1982). The receptor for transferrin and the phosphomannosyl receptor also are found clustered in coated pits in the absence of exogenously added ligand (Willingham et al., 1983b, 1984). Presumably this clustering is related to the fact that these receptors continuously recycle in order to bring metabolically
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important substances such as cholesterol (LDL), iron (transferrin), or newly synthesized lysosomal components into the cell. On the other hand, the epidermal growth factor (EGF) receptor of KB cells has been shown to cluster in coated pits only after the addition of ligand. In addition, this clustering is temperature dependent. It is unable to do so at 4°C in spite of the fact that it is mobile in the membrane at this temperature (Hillman and Schlessinger, 1982; Willingham and Pastan, 1982). When the binding characteristics for a,M were analyzed using radiolabeled material, the binding affinities indicate that there are two classes of receptors (Dickson et al., 1981b; Hanover et al., 1984). The high-affinity class of receptors is detected under conditions in which the ligand-receptor complexes are found clustered in coated pits on the cell surface. This finding has led to the suggestion that there may be some conformational change in a,M receptors that results in both a higher affinity for a2M and accumulation in coated pits. In order for different types of receptors to become clustered in the same coated pit, receptors should share some common structural feature that enables a recognition system in the pit to selectively concentrate them prior to internalization. No protein structural data to indicate the nature of this common feature is available at this time. The number of receptors on the plasma membrane is often, but not always, regulated by occupancy. For example, the number of receptors for a,M and transferrin are relatively constant under various culture conditions. On the other hand, the number of the receptors for LDL, EGF, and insulin are lowered or down-regulated by the presence of LDL, EGF, or insulin, respectively. Since serum contains LDL, exposure of cells to LDL-deficient medium for a few days is necessary to increase the number of LDL receptors (Goldstein et al., 1979). Receptors are not always uniformly distributed over the cell surface. In hepatocytes, the cells are separated by junctional elements such that only one side of each cell has receptors for the asialoglycoprotein receptor (Wall et al., 1980). In a similar fashion, the upper and lower cell surfaces of cell lines like MDCK, which have junctional complexes, have been found to contain different components (Rodriquez-Boulan and Pendergast, 1980). Thus, cells can regulate the total number of receptors on their surface, and the portion of the surface on which they are located. Some cell types have domains of their membranes which have higher concentrations of receptors than adjacent areas in which junctional complexes have no role. For example, microvilli have been reported to have higher receptor numbers than adjacent areas of membrane (Weller, 1974). When HCG is added to granulosa cells, the ligand bound to its receptor appears to have an extremely long residence time on the cell surface (8 hours) (Ahmed et al., 1981). Because many components of the cell surface are internalized every few hours, this unusual receptor system probably may have a special mechanism for keeping it on the cell surface.
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B. CLUSTERING IN COATED PITS The entry of ligand-receptor complexes into the specific endocytic pathway from the cell surface is mediated by a unique structure termed a coated pit which is present on most animal cells. In many cultured mammalian cells there are 500-1500 coated pits on the surface of each cell. These structures serve as foci in which receptor-ligand complexes are concentrated just prior to internalization. In this manner a cell can selectively concentrate ligands from other parts of the cell surface and internalize them, without internalizing other portions of the plasma membrane. Coated pits at the plasma membrane are relatively uniform in size in any one cell type (100-150 nm across). They derive their name from the fuzzy bristle coat that can be seen by transmission electron microscopy on the cytoplasmic face of the plasma membrane in these regions. They have been postulated to have a specific association with actin-containing microfilament bundles in cultured cells because they have been noted to be aligned along stress fibers in flat cells (Anderson et al., 1980; Salisbury et al., 1980). However, this association with stress fibers is more likely related to overall cell organization than to specific interactions of coated pits with microfilament bundles (Willingham et al., 1979, 1981e). Coated pits involved in endocytosis were probably first observed in erythroblastic bone marrow cells by Bessis alid co-workers (Bessis, 1963; Bessis and Breton-Gorius, 1962; Policard and Bessis, 1958), but the nature of the coat of these pits was not recognized. It was subsequently demonstrated by Fawcett (1964) that the pits described by Bessis were actually “coated” pits. In 1961, Gray published images of the coat structures in neuronal cells (Gray, 1961), although he later questioned their nature (Gray, 1972). Between 1962 and 1964, a number of reports demonstrated the existence of coated structures in diverse cell types such as liver (Ashford and Porter, 1962; Rouiller and Jezequel, 1963), insect oocytes (Anderson, 1964; Roth and Porter, 1962, 1964), other insect cells (Bowers, 1964), kidney (Wissig, 1962), and neuronal cells (Brightman, 1962; Brightman and Palay, 1963). The possible role of these structures in endocytic activity was repeatedly discussed. Roth and Porter (1962, 1964) coined the term “bristle-coated pits”; this terminology has been widely used. The concentration of a specific ligand in coated pits was first demonstrated using labeled LDL on cultured fibroblasts (Anderson et al., 1976). Subsequently, many ligands have been found to be concentrated in coated pits in studies on a wide variety of cultured cells and in intact animals. The process of clustering in coated pits varies in detail from receptor to receptor. Some receptors, such as those for EGF, are diffusely distributed when unoccupied, and cluster in coated pits only after addition of ligand (Willingham and Pastan, 1982). LDL receptors, as mentioned above, appear to be able to cluster even in the absence of ligand (Anderson et al., 1982). Clustering of the
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EGF-EGF receptor complex in coated pits does not occur at 4°C; higher temperatures are required for clustering to occur (Willingham and Pastan, 1982). This inhibition of clustering does not appear to be due to restricted lateral mobility of the EGF receptor (Hillman and Schlessinger, 1982). A number of chemical substances have been found to inhibit clustering in coated pits (Cheng ef al., 1980; Davies et al., 1980; Dickson et al., 1981b, 1982b,c; FitzGerald et al., 1980; Haigler et al., 1980a,b; Hanover et al., 1983; Levitzki et al., 1980; Maxfield et al., 1979a; Schlegel et al., 1982a; Via et al., 1982). One of the most potent of these inhibitors is dansylcadaverine. Because dansylcadaverine is a potent inhibitor of the enzyme transglutaminase, it was suggested that this enzyme might have a role in the clustering process (Davies et al., 1980). Recently, dansylcadaverine has also been found to be a potent inhibitor of phosphatidylcholine biosynthesis (Mato et al., 1983) and to stimulate phosphotidylinositol synthesis. It is hoped that studies with compounds that block clustering in coated pits may help clarify the biochemical mechanism that regulates the clustering process. One area that has been overlooked in considerations of the coated pit pathway is its contribution to nonconcentrative endocytosis, both adsorptive and fluidphase. Some of the confusion has come from experiments in which a marker, such as horseradish peroxidase (HRP), that had been used in one cell type (such as macrophages) to identify the major pathway of fluid-phase endocytosis, was used in a different cell type (such as cultured fibroblasts) and labeled a different organelle. Macrophages and fibroblasts differ significantly in their surface ruffle and macropinocytotic activities. When cultured fibroblasts are incubated at 4°C with HRP, there is no specific binding of HRP to the plasma membrane; thus, it is not an adsorptive marker. However, when the incubation is conducted at 37"C, a large amount of HRP is rapidly found in the same endocytic vesicles that contain ligands that, in double-label experiments, are found bound to the cell surface and concentrated in coated pits prior to entry (Ryser et al., 1982; Willingham and Pastan, unpublished data). Thus, we can conclude that in such cells HRP enters via coated pits, whereas in macrophages it may mainly enter in macropinosomes. The question that often arises is how much of the observed uptake is fluid-phase through the coated pit system, and how much is adsorptive through the coated pit system. One striking fact that is clear from quantitative studies on ligand uptake is that internalization by the coated pit system can be very rapid. Estimates from quantitative ligand uptake suggest that each coated pit may be able to cluster ligands and produce an endocytic vesicle every 20 seconds at 37°C (Pastan and Willingham, 1981b). The large amount of fluid taken up rapidly by this pathway indicates the slower processes of macropinosomal and caveolae vesicle formation are probably much less important in fluid uptake in these types of cultured cells than previously thought.
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C. THE STRUCTURE OF COATED PITS:CLATHRIN Following the morphologic description of coated pits as structures in fixed cells by electron microscopy, coated vesicles were isolated from cell homogenates and subjected to biochemical analysis. Pearse (1975, 1976, 1978) identified the major protein component of coated vesicle preparations as a protein with a M, of 180,000, which formed a basket-like coat around the vesicle. She named this protein “clathrin,” from the Latin “clathra-” meaning “lattice.” Subsequent work showed that this protein could be purified and induced to form basket-like coat structures in the absence of membranes (Pearse, 1978; Fine et al., 1978; Keen et al., 1979; Woodward and Roth, 1978). The structure of the clathrin coat is rather unique in that it has the appearance of a latticework arranged as hexagons and pentagons similar to the surface of a soccer ball (Crowther and Pearse, 1981; Crowther et al., 1976; Kanaseki and Kadota, 1969; Kirchhausen and Harrison, 1981; Schook et al., 1979; Ungewickell and Branton, 1981). These basket structures have been shown to contain proteins in addition to clathrin and these other proteins may also be required for the integrity of the basket structures seen in the living cell (Kirchhausen and Harrison, 1981; Ungewickell and Branton, 1981; Pearse, 1978; Crowther and Pearse, 1981). Antibodies to clathrin have been produced and used to analyze the location of clathrin in intact cells (e.g., Willingham et al., 1981b). Many, if not all, of these antibodies are directed against one or both of the low-molecular-weight proteins that associate with the 180 kilodalton (kd) clathrin molecule (Brodsky and Parham, 1982; Keen et d., 1981; N. D. Richert, M. C. Willingham and I. Pastan, unpublished data). The components of coated vesicles have been referred to as the clathrin heavy chain (1 80 kd) and clathrin light chains (LC-A and LC-B, 36 and 33 kd) (Kirchhausen and Harrison, 1981; Pearse, 1978). In cultured fibroblasts the clathrin distribution determined by immunocytochemistry corresponds exactly to the distribution of morphologically visible coated structures seen by transmission electron microscopy. Immunocytochemical studies at the ultrastmctural level have also suggested that very little, if any, clathrin is present in other sites (Willingham el al., 1981b). In cultured fibroblasts and epithelial cells there are two populations of clathrincoated structures: one group of coated pits (approx. 150 nm diameter) is associated with the plasma membrane (about 1000 per cell). The other group of coated pits is smaller (approx. 70 nm in diameter) and found in the transreticular elements of the Golgi system (about 200 per cell). Both of these clathrin-coated structures react with antibodies made against clathrin prepared from mammalian brain (rat, bovine, or human). When tissues are homogenized, they yield two populations of closed vesicles which differ in their size. The predominant class obtained from homogenization of brain is the smaller (70 nm) size class. When the coat lattice is investigated by
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transmission electron microscopy, it appears in sections as a periodic, spikelike array covering the cytoplasmic face of these structures (Fig. 2 ) . When isolated baskets are studied by negative staining or shadowing methods or observed in tangential section in intact cells, one can see the hexagon-pentagon arrangement of the basket. These hexagon-pentagon arrangements are shown in very clear images using cell “fracture” and surface replica techniques (Heuser and Evans, 1980). D. VESICLEFORMATION IN ENDOCYTOSIS FROM COATEDPITS
1 . Coated Vesicles? While coated pits serve as foci of concentration of receptor-ligand complexes on the cell surface, there is disagreement about whether the coated structures themselves ever leave or pinch off from the plasma membrane during the endocytic process. Endocytosis via coated pits occurs very frequently (about every 20 seconds in fibroblasts) and the endocytic event (the transfer of ligand from coated pits to uncoated vesicles within the cell) probably occurs within a few seconds or less at 37°C. It has been assumed that during endocytosis the coated pit pinches off from the plasma membrane to form an isolated coated vesicle, but direct evidence that this occurs in living cells is lacking. The evidence which suggested that these coated pits do pinch off to form coated vesicles is mainly based on two types of experiments. The first is that one can retrieve from cell homogenates intact isolated vesicles bearing a clathrin coat (coated vesicles) (Pearse, 1976). The second is that in electron microscopic images one can see cross sections of vesicular structures bearing a clathrin coat (Roth and Porter, 1964; Friend and Farquhar, 1967; Franke et al., 1976; Anderson et al., 1976). In addition, biochemical characteristics of clathrin indicate that it is able to assemble and disassemble in a test tube with sufficient ease that an assembly process seemed possible in the living cell (Keen et al., 1979). There are some difficulties, however, with these lines of evidence. One problem is that the existence of isolated vesicles after homogenization does not necessarily indicate that the structures existed as isolated vesicles in the living cell. The vesiculation of membrane structures by homogenization is well known for microsomes derived from endoplasmic reticulum or for small plasma membrane vesicles derived from intact plasma membrane. Further, observation of isolated vesicle cross-sections by electron microscopy in section does not prove that these structures are actually isolated (Fig. 2 ) . A glancing section of a structure with a tortuous shape can yield this same image without the structure being isolated from other membranous connections. It has been shown previously that most of the structures that appear to be isolated vesicles in section are, in fact, connected to the cell surface, because they can be heavily labeled by impermeant
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FIG.2. Morphologic appearance of clathrin-coated pits at the plasma membrane. K B human carcinoma cells (A-K) or Swiss 3T3 mouse fibroblast cells (L-0) were fixed and processed using glutaraldehyde and routine Os04 (F,G,H,I,J,K,), ferrocyanide-reduced Os04 in the OTO sequence (Willingham and Pastan, 1983a) (L,M,N,O), or routine Os04 in the OTO sequence (Willingham and Rutherford, 1984). The typical appearance of clathrin-coated pits is shown in (A-C). (E) shows two different shapes of the coated regions (arrowheads). (B,F) show the appearance of “cryptic” pits
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markers applied to the cell exterior after cell fixation (Willingham et al., 1981d). This type of study clearly shows that the majority of the plasma membrane coated structures are not isolated but are connected to the cell surface. Other experiments with impermeant markers have shown that the communication of these structures with the cell surface is a temperature-dependent process. Virtually all of these large surface-related coated structures can be shown to communicate with the cell surface at 4°C (Willingham et al., 1981d). However, at 37°C a significant percentage (about 30%) of plasma membrane-type coated structures label poorly with impermeant markers after fixation (Wall et al., 1980; Willingham et al., 1981d). These studies also showed that one could find images of coated pits with narrow necks whose membranes were continuous with the cell surface membrane at 37”C, but failed to admit the impermeant marker (cryptic coated pits; Fig. 2). This has led to the question of whether these colloidal impermeant markers, such as ruthenium red (Wall et al., 1980; Willingham er al., 1981d), lathanum hydroxide (Shaklai and Tavassoli, 1982), lathanum carbonate (M. C. Willingham, unpublished data), tannic acid (Blanchette-Mackie and Scow, 1982; M. C. Willingham, unpublished data), or horseradish peroxidase (Willingham et al., 198 Id) can unequivocally prove the lack of anatomical communication of one structure with another structure. If coated pits pinch off to form coated vesicles, the clathrin must return to the cell surface. This presumably would happen by the coat disassembling and the “soluble” disassembled clathrin returning to the surface. Using antibodies to clathrin, immunocytochemical methods have been employed to determine the (Willingham et al.. 1981d) which exclude impermeant extracellular marker (Ruthenium Red), in spite of their continuity with the plasma membrane. (G-K) are cells incubated with lathanum chloride after fixation in glutaraldehyde, followed by rapid washing in sodium carbonate to produce an impermeant precipitate of lathanum carbonate. (G) demonstrates an image showing the narrow neck connection of a cryptic coated pit (arrowhead) with the cell surface; note the sudden decrease in accessibility to the impermeant marker at the upper edge of the coated region, which admits only small amounts of the marker beyond the restriction (arrows). (H-K) show varying planes of section of pits that show communication with the surface by their content of lathanum label (arrows). (H) shows membrane connection to the surface, which become more and more tangential to the surface connection in (ILK), until in (K) the vesicular profile has the appearance of an isolated vesicle. The presence of lathanum precipitate in (K), however, shows that this structure has a connection to the cell surface that is still intact, since the lathanum was added only after fixation with glutaraldehyde. The lack of labeling with such impermeant markers does not prove that the vesicular structure is not connected to the cell surface, as shown for the cryptic pits shown in (B,F,G). (L and M) are adjacent serial sections of the same coated pit labeled with concanavalin A-horseradish peroxidase (con AHRP). The vesicular image in (L) (arrowhead) can be seen to be, in reality, a narrow-necked pit in connection with the surface in (M). (N and 0) show similar narrow-necked structures during the endocytosis of Con A-HRP. In these images, the narrow necks (arrows) connect the coated pits (arrowheads) to the cell surface. (A,B,C, x 140,000; D, x240,OOO; E,F,H,I,J,K,N,O, x90,OOO; G , X 138,000; L,M, X 180,000; bars = 0. I pm; lead citrate counterstain.)
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location of clathrin. The only structures in the cell that showed significant amounts of clathrin morphologically were coated pits (Willingham et al., 1981b). A significant pool of diffusely distributed “soluble” clathrin could not be found (Willingham et al., 1981b; Wehland et al., 1981). A series of direct experiments have also been performed to investigate how uncoated vesicles form from coated pits. Using microinjection to introduce antibodies against clathrin into living cells, it could be shown that the antibody gained access to all of the cytosolic regions of the cell, and that it reacted with the exposed clathrin on the cytosol face of all pits (Wehland et al., 1981). However, precipitation of clathrin aggregates or isolated coated vesicles in the cytosol was not seen even after the antibody was present for many hours. In addition, the process of endocytosis from these coated pits proceeded normally in spite of their decoration by anticlathrin antibody. This antibody was active in the sense that it could precipitate soluble clathrin or isolated vesicles from an exogenous source when these were injected at a later time into cells previously injected with anticlathrin antibody. More recently, studies have appeared which utilized seriaf sections of surface coated pits in intact cells to investigate if images of coated vesicles are connected to the cell surface (Fan et al., 1982; Peterson and van Deurs, 1983). These authors have reported that from 10 to 50% of coated vesicular profiles in different cell types seemed to be isolated coated vesicles when adjacent serial sections were examined. One difficulty with such studies is that the density of membranes is very close to that of the surrounding cytoplasm, particularly when these membranes are seen in tangential section in very small tubular structures (700 nm in length) and have tortuous shapes. We have compared our method with the preservation-contrast techniques used in previous studies and find that these other methods fail to preserve the narrow necks. We feel that this new morphologic study, taken with the previous evidence from immunocytochemical, impermeant marker, and microinjection studies, strongly argue that coated pits do not pinch off to form isolated coated vesicles during the endocytic event. How we think this step in the endocytosis process might occur will be discussed below.
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2. Receptosomes When a,M is labeled with rhodamine, its uptake into living cells can be observed by fluorescence image intensification microscopy (Pastan et al., 1977; Willingham and Pastan, 1978, 1980, 1983e). The clustering of ligands on the surface of cells is difficult to see clearly because of the large amount of diffuse binding over the entire surface of the cell, and the quenching of the rhodamine signal that occurs when this directly labeled ligand is clustered in coated pits on the cell surface (Willingham et al., 1981a). However, after only a few minutes at 37°C the pattern of fluorescence changes, and small rapidly moving punctate dots appear within the cell (Pastan and Willingham, 1981b). These structures are vesicles which move by saltatory motion, a characteristic form of motion generally restricted to intracellular organelles such as mitochondria and lysosomes (Freed and Lebowitz, 1970; Rebhun, 1972; Wehland and Willingham, 1983; Willingham et al., 1979; Willingham and Yamada, 1978). These fluorescent structures are not visible by phase contrast microscopy, which indicates that they are either very small and/or they are not very dense. Electron microscopy shows that both of these predictions are correct (Willingham and Pastan, 1980). These organelles show the unusual property, in contrast to macropinosomal endocytic vesicles (Lewis, 1931; Straus, 1964), of not fusing with lysosomes when they approach them as they move along microtubule tracks used for saltatory motion. These structures are, therefore, special nonlysosomal vesicles which move by saltatory motion and which contain ligands that have entered cells via coated pits. They were named “receptosomes” to emphasize their role in the uptake of receptor-mediated ligands (Willingham and Pastan, 1980). Typical examples of the appearance of these vesicles are shown by electron microscopy in Fig. 3; their characteristics are listed in Table I. They have appeared in electron micrographs for many years, but it was not always known where they came from or what their destination might be. They have been variously identified as condensing vacuoles near the Golgi, tubulo-vesicles, endosomes, intermediate vacuoles, pinocytic vesicles, multivesicular bodies (presumed to be lysosomal), CURL, mature lysosomes, macropinosomes, phagocytic vacuoles, or simply as lucent vesicles or vacuoles (Abrahamson et al., 1979; Abrahamson and Rodewald, 1981; Ackerman and Wolken, 1980, 1981; Ackerman et al., 1983; Anderson et al., 1976, 1977, 1982; Bergeron et al., 1979; Bessis, 1963; Bleil and Bretscher, 1982; Bretscher et al., 1980; Brown et al., 1982, 1983; Carpentier et al., 1978, 1979a,b, 1982; Courtoy et al., 1982; Dales, 1973, 1978; DeDuve and Wattiaux, 1966; Fan et al., 1983; Farquhar, 1978, 1981, 1983; Franke et al., 1976; Friend and Farquhar, 1967; Geuze et al., 1983; Gonatas et al., 1977, 1980; Gorden et al., 1978a,b; Haigler et al.. 1978, 1979; Handley et al., 1981; Helenius et al., 1980; Helenius and Marsh, 1982; Herzog and Farquhar, 1977; Herzog and Reggio, 1980; Iacopetta et al., 1983;
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FIG.3 . Morphologic appearance of receptosomes. Swiss 3T3 cells (A,B) or K B human carcinoma cells (C-Q) were fixed in glutaraldehyde and processed using ferrocyanide-reduced OTO (A,B), routine OsO, (C), or routine OsO, OTO (Willingham and Rutherford, 1984) (D-Q). Receptosomes were labeled with Con A-HRP (B), epidermal growth factor-horseradish peroxidase (C), or unlabeled (A,D-Q). The appearance of receptosomes initially after formation is shown in (A-E), in which they characteristically appear empty, and often contain a single intraluminal vesicular profile (small arrows), as well as a fuzzy decoration on a single straightened edge of their cytoplasmic
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TABLE I PROPERTIES OF RECEPTOSOMtS Empty appearance by electron microscopy Move by saltatory motion Contain ligand derived from plasma membrane coated pits Selectively fuse with trans-reticular Golgi Do not directly fuse with lysosomes Do not contain lysosomal hydrolases Have an acidic pH (approx. pH 5.0) Have a smooth, continuous membrane in routine EM processing (lysosomes have a fragmented membrane) Often 10% of membrane is straightened and contains a fuzzy fibrillar material on its cytosolic face Often contain a single intraluminal vesicular structure (forms when receptosomes form) Fuse with other receptosomes creating multivesicular receptosomes The isolated vesicle has an intermediate bouyant density between plasma membrane and lysosomes Often contain ligands and receptors
Joseph et al., 1978, 1979; Marsh and Helenius, 1980; M a t h et al., 1982; McGookey et al., 1983; Michaels and Leblond, 1976; Nagura et al., 1979; Nicolson, 1974; Novikoff et al., 1980; Orci et al., 1978; Ottosen et al., 1980; Posner et al., 1981; Quintart et al., 1983; Rodewald, 1973; Rodewald and Abrahamson, 1980; Ryser et al., 1982; Salisbury et al., 1980; Silverstein et al., 1976; Simons et al., 1982; Steinman et al., 1983; Straus, 1964; Wall et al., 1980). In many cases the relationship of these structures to endocytosis was not recognized. In any random section of cells fixed from 37"C, the frequency with which images of receptosomes appear is low; this probably reflects their short life span in cells (often less than 10 minutes). This, plus their relatively nondescript appearance, probably led to the failure to describe them as specific organelles. surface (arrowheads). This f u z z y decoration has a substructure which appears lamellar and periodic, shown at higher magnification in (G,H). With OTO fixation, one can also see the densities of lipoprotein particles contained within these receptosomes (small arrowheads-F,I,K,M,O), presumably representing low-density lipoproteins taken up from the culture medium. At later times, receptosomes fuse with each other, generating larger receptosomes that contain multiple intraluminal vesicular profiles (small arrows-F,I,J,K) and multiple fuzzy edges (large arrows-H,I,K,M). A commonly observed fixation artifact in these vesicles (caused by glutaraldehyde) is the inward blistering on the membrane, producing a C-shaped profile (large arrows-L-Q). The artifactual nature of these blisters is evident from the empty appearance of the central regions of the blisters, indicating a lack of cytosol matrix material and, therefore, formation after the cytoplasmic matrix had been stabilized by glutaraldehyde fixation. One of these blisters is shown in cross section in (N), making it appear to be a separate vesicle contained within the receptosome. (A-F, 1-Q, X90,OOO: G,H, X 180,000; bars = 0.1 pm; lead citrate counterstain.)
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Ligands which bind to specific receptors can usually be detected concentrated in coated pits prior to entry into receptosomes, but as discussed above other substances which do not concentrate in pits also enter the cell through this common endocytic pathway. For example, horseradish peroxidase does not bind to specific receptors on the cell surface of fibroblasts and is not found concentrated in open coated pits. Yet 10 minutes after cells are incubated with HRP the ligand is found in typical receptosomes (Fig. 3) (Ryser et al., 1982; Willingham and Pastan, unpublished data). Because, as far as is known, receptosomes only form from coated pits, this experiment indicates HRP enters cells through coated pits, but in an unconcentrated form.
111. Endocytic Vesicles: Characteristics
A. RECEPTOSOMES The general properties of receptosomes and their identification as vesicles derived from coated pits at the plasma membrane were described in the previous section. In this section, the morphologic characteristics of endocytic organelles will be discussed. Receptosomes can be described as clear vesicles, since they generally have very little electron-dense material in their interior (Fig. 3). Their small size and low density make them invisible by light microscopic methods that depend on refractile resolution, such as phase contrast microscopy. They generally contain a very small single intralumenal vesicle when they first form from coated pits. It is possible that some of these intralumenal vesicles represent images of infoldings of the receptosomal membrane seen in tangential section. Receptosomes have been observed to fuse with each other in living cells when observed by video intensification microscopy (Willingham and Pastan, 1983b). As a result, at later stages they can become multivesicular (Fig. 3 ) and represent one class of vesicles that have been termed “multivesicular bodies.” (However, the term multivesicular body is a “catch-all” phrase used to describe many different organelles.) In some cultured cells, such as very flat fibroblasts, there is a considerable separation of the peripheral cytoplasmic surface from the cell center where most of the elements of the Golgi system are located. In such cells, receptosomes that form from coated pits in the peripheral parts of the cell may travel 10-100 p m to fuse with elements of the Golgi. It is in such cells (such as Swiss 3T3 cells) that receptosomes are easiest to visualize and study, since their life span can be from 15 to 60 minutes depending on the part of the cell from which they originate. On the other hand, in more compact or rounded cells, such as KB cells or CHO cells, there is often a very short distance ( I , e, increases without limit and error catastrophe is inevitable. Clearly, in the framework of this model any cell which is not rapidly to become extinct must have a qr- I , then it must intersect the line qr = qr- I in two points and the higher of these defines a point of stable accuracy, qstable.The lower point of intersection, qthreahold, is also a point of constant accuracy, but it may readily be seen that it is unstable. Provided accuracy does not at any time fall below qthreshold it will always return to qstable after a perturbation, but if accuracy does drop below qthreshold it will thereafter decline irreversibly toward error catastrophe. The increased versatility of the adaptor model compared with Eq. (1) is readily seen if Eq. ( 1 ) is plotted in a form similar to Fig. 2 (see Fig. 3). In this case, the relationship between qr and qt- I is a straight line which either crosses (a< 1) or does not cross (a >1) the line of constant accuracy. Because a is treated as a constant, independent of q, stability is either absolute, or nonexistent. No threshold level of accuracy exists for Eq. ( I ) , so the transition of a cell from stability to instability is either disallowed, or requires a change in the parameter a. The flexibility of the adaptor model is further increased if account is taken of the stochastic nature of errors in protein synthesis. Because the deterministic model is much more readily tractable than its stochastic counterpart, Eq. ( 2 ) has been derived only in the deterministic case. In reality, however, the expression for qr on the right-hand side of the equation represents only some form of statistical average, about which there will at any time be random scatter (see
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qt
1.o
lfh qt-1
FIG.2. Relationship between q, and q r - , predicted by adaptor model of error propagation (Hoffmann, 1974; Kirkwood and Holliday, 1975). The curve intersects the line q, = q r - , in two points, the higher of which defines a point of stable accuracy, qrt&le. Provided accuracy remains above the lower point of intersection, qthreshold, it will always revert to its stable value. Below qthreshold, accuracy falls progressively resulting in total loss of specificity (9 = ]/A, where A is the number of different amino acids). Separation between qstahle and qthreshold is determined by two parameters, S (specificity of error-free adaptors) and R (residual activity of erroneous adaptors); see text for definitions. Increasing S moves the curve upward, including the right-hand endpoint. Decreasing R moves the curve upward and to the left, leaving both endpoints fixed. It is possible also for the curve to lie entirely below the line q, = 4,- , in which case no point of stable accuracy exists (not shown).
,
Hoffmann, 1974; Kirkwood and Holliday, 1975a). By considering the probabilities of correct and incorrect insertion of the n residues as well as the rates, an approximation to the standard error of qr may be written as
where N is the number of viable (i.e., normal + erroneous) adaptors present (Kirkwood and Holliday , 1975a). If the separation between qstableand qthreshold is not too large relative to SE (q),there will be a possibility of a purely random transition from the stable to the unstable state. One further model of error propagation in protein synthesis was described by
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0
Frc. 3. Relationship between qr and q,-, for a model with linear feedback of errors (Orgel, 1970). Depending on whether the linear feedback coefficient a is less than or greater than one, translation is either always stable or always unstable. No threshold level of accuracy exists for this model.
Goel and Ycas (1975). However, it has been argued elsewhere that this model is fundamentally flawed (Kirkwood, 1980) so we shall not consider it further here.
B. GENERALIZED THEORY Models based on protein synthesis alone demonstrate certain principal features of error propagation in a self-replicating translation apparatus. However, as has been remarked already, a fully realistic theory will have to take account of transcription and DNA replication errors as well. Since proteins are involved in each of these operations, the possibilities for highly complex feedback relationships are many, and to date no detailed mathematical model involving multiple levels of information transfer has been described. Kirkwood (1980) reviewed in outline the primary features which a more general model will require, and these are represented diagrammatically in Fig. 4. More recently, Hasegawa et al. (1984) reconsidered these points in relation to evolution of subcellular organelles and suggested a numerical classification of errors into three types. Type I errors are the errors which arise simply in the
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translation step of protein synthesis. Type 2 errors are errors in the transcription of DNA to RNA. Type 3 errors are mistakes in the replication or repair of DNA, or in other words, gene mutations. It is also useful in thinking about the dynamics of error propagation to restrict attention only to those constituents of DNA, RNA, and protein that are themselves involved in some aspect of the translation process. With this restriction, we are interested only in how errors are propagated within the process itself, and we can defer for a moment consideration of how the rest of the cell’s functions are affected. Type 3 errors are qualitatively different from types 1 and 2 in that, once made, they are permanent, whereas types 1 and 2 last individually only as long as the protein or mRNA molecule persists within the cell. In consequence, type 3 errors are very much more serious, and it is unsurprising that they occur at very much lower rates (see below). Type 2 errors are again more serious than type 1 since, on average, a single message may be translated many times and, therefore, an error in transcription will tend to produce a burst of errors in newly synthesized protein. On the other hand, type 1 errors although individually less serious are likely on average to be by far the most numerous and it is therefore reasonable to regard type 1 errors as the primary medium through which error propagation takes place. The differences in frequency and effect of types 1 and 2 errors may be of particular significance in the way a stable translation process could become
I
I
Type3
DNA polymerase
1
I -1,
- DNA
I
I d
Ribosomal proteins, aminoacyl-tRNA synthetases
Protein
FIG. 4. Schematic representation of the main pathways of information transfer between macromolecules. The bold arrows denote transcription of DNA into RNA and translation of RNA into protein. The dashed arrow denotes DNA replication. The remaining arrows indicate pathways for cyclic propagation of errors through mistakes in the synthesis of information-handling proteins.
T. B. L. KIRKWOOD ET AL
I02
I
Error catastrope
Error level
I
Tvoe 2 errors
Time FIG.5 . The error level in the cellular translation process is expected to fluctuate randomly about qatableunless it crosses qthresholdwhen it will increase progressively to error catastrophe. Type 2 (transcription) errors might be particularly significant in destabilizing the translation process since a single mistake will produce many erroneous proteins. In the case illustrated, two type 2 errors are assumed to have occurred in close succession.
destabilized. Periodically, a type 2 error might cause a relatively large increase in the protein synthetic error rate and this might on occasion be sufficient to push the system past the threshold of recovery (Fig. 5). Once destabilization had occurred, the error frequency would continue to rise under the impetus primarily of type 1 errors, until eventually the accuracy of synthesis of RNA and DNA polymerases became so low that types 2 and 3 errors would occur more frequently, and these would then contribute to the final demise of the cell (Orgel, 1973; Lewis and Holliday, 1970; Fulder and Holliday, 1975; Kirkwood, 1980). The scenario in which cells experience an initial build up of protein errors, followed by progressive deleterious changes in RNA, DNA, membranes, organelles, and so on, is the basis of what is termed the general error theory of aging (Holliday and Kirkwood, 1983). [This theory should not be confused with the somatic mutation theory (Szilard, 1959; Curtis, 1966; Burnet, 1974; Holliday and Kirkwood, 198'1; Morley, 1982) which attributes cell aging directly to the gradual accumulation of multiple mutations in the genetic material of cells, and which does not invoke the idea of error propagation (Holliday and Kirkwood, 1983). In contrast, the error theory predicts gene mutations will occur, but does
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not require them, and a key point is that even with a mutation-free genome a cell cannot survive if either it inherits a nonviable translation apparatus or the accuracy of its translation breaks down. J
111. Evolutionary Considerations A. ORIGINS OF TRANSLATION The evolutionary origins of genetic translation remain obscure (Crick et af., 1976; Miller and Orgel, 1973; Eigen and Schuster, 1979). Various models have been proposed based on properties of the genetic code (Crick, 1968; Woese, 1967; Hartman, 1975), while others have developed from the assumption that there existed an initially random autocatalytic system comprising polypeptides and polynucleotides (Hoffmann, 1974; Mizutani and Ponnamperuma, 1977). Whatever the true sequence of events may have been, at some point primitive replicators acquired the ability to separate the responsibilities of storing and implementing genetic information among two different sets of molecules and thereby gained a greatly enhanced adaptational flexibility. For the purpose of this review, we ignore the steps leading to the first instance of genetic translation and consider only the selective pressures which operated subsequently. B . SELECTIONFOR ACCURACY Since the translation process is fundamental to every aspect of life, it is obvious that natural selection acts as readily on the accuracy of macromolecular synthesis as on any other trait. The force of this selection may be judged from the impressive array of mechanisms which cooperate to produce the high fidelity of synthesis of DNA, RNA, and proteins that is found in present-day species. The primitive translation process presumably lacked anything but the most basic chemical or mineral catalysts and it would have depended primarily on simple differences in binding energies and stereochemistry for its specificity of translation. Gradually, there must have evolved enzymes, which were themselves the products of translation, that had greater and greater specificity until at some point the entire replication and translation machinery became fully autocatalytic. In the evolution of the translation process from its primitive state to the complex system we see today, natural selection would have favored two separate, but related characteristics: accuracy and stability. The selection pressure continually to improve these characteristics may be understood in terms of the deleterious consequences of errors in RNA or protein synthesis. These errors have both a general consequence and a specific one. The general consequence is a lowering of metabolic efficiency: it is a waste using energy and resources to
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synthesize an RNA or protein molecule which cannot perform its normal function. For this reason alone, we can expect selection to produce a high degree of translational accuracy. The specific consequence relates only to the proteins required for information transfer between macromolecules: it is the possibility of error propagation which could lead ultimately to cell death. As discussed earlier, error propagation occurs initially in proteins, but in due course it will also affect the fidelity of DNA replication itself. Therefore to protect its DNA, the organism must achieve a considerable degree of accuracy in transcription and translation, and it must also ensure that the translation process as a whole remains stable. The evolution toward greater translational accuracy and stability can be described most conveniently in the framework of the Hoffmann-Kirkwood-Holliday (HKH) model, described in the previous section (see Fig. 2). By selecting for greater specificity, S , the curve in the plot of q, against 4,- is raised. This results in an increase in the average accuracy, q\table.By selecting for lower residual activity, R , the curve is raised upward and to the left in such a way that the endpoints remain fixed. This also results in an increase in qctable,but less directly. Both these changes are likely also to increase the separation between qstable and qthre+,ld and therefore to decrease the risk of precipitating the cell onto the path to error catastrophe. Evolution toward reduced R is most readily seen as occurring through acquisition of scavenging mechanisms for preferentially degrading erroneous protein, although in the early evolution of enzyme structures there may also have been selection for information handling molecules which have stringent requirements for structural stability and which are therefore likely to be highly labile if an error is made in their synthesis. The benefits of raising the accuracy and stability of translation are obvious, but it is also clear that there are costs. The cost may either be a direct cost, in the sense of an increased energy requirement, or it may be an indirect trade-off against, for example, the rate of protein synthesis. Generalized schemes for proofreading the synthesis of macromolecules have been discussed by Hopfield (1974) and by Ninio (1975). In Hopfield’s kinetic proofreading scheme, the accuracy of synthesis is raised by means of one or more intermediate reaction steps in which the enzyme-substrate complex is driven into a high-energy transitional state from which there is an increased probability of dissociation of an incorrect match. In Ninio’s “delayed reaction” scheme, each step of synthesis is subjected to a time delay, during which the differential likelihood of dissociating correct and incorrect bonds results in an increased level of discrimination. This causes a slowing of the total rate of synthesis, and it also increases the chance that correct bonds may dissociate, requiring reinitiation of the entire step. Other mechanisms, such as “double-sieve’’ editing (Fersht and Dingwall, 1979) require that, on average, more than one unit of substrate (amino or nucleic acid) is inserted and excised during each completed step, and these consume both energy and time. Similarly, a variety of costs can be identified in the development of
,
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protein scavenging systems, where preferential elimination of abnormal molecules is likely to involve a greater turnover of normal protein as well, and there is experimental evidence that a cell’s ability to hydrolyse abnormal protein does indeed require energy (Goldberg, 1972; Bukhari and Zipser, 1973; Waxman and Goldberg, 1982). Theoretical analysis by Savageau and Freter ( 1 979) of the energy cost of proofreading the aminoacylation of tRNAs concluded that this alone accounts for approximately 2% of the energy required to synthesize a bacterial cell. In rapidly growing bacteria, it has been shown that ribosomes account for up to 50% of the RNA and 30% of the protein which is synthesized (Maaloe, 1979), and since evolution of the ribosome is likely to have been directly related to the evolution of accurate translation, this too represents a major investment in accuracy. Therefore, although detailed calculation of the full metabolic investment in accuracy is not yet possible, it appears highly probable that it is a very significant fraction of a cell’s total energy budget. The upshot of these considerations is that while a high level of translational accuracy and stability is clearly of positive benefit to the organism, there is likely to be a law of diminishing return, such that the costs of raising S and reducing R both increase sharply. In primitive organisms, when the fidelity of translation was presumably low, the benefit of increased efficiency and stability resulting from increased accuracy would initially have outweighed its cost. This principle might be expected to have resulted in a high degree of translational stability, especially in unicellular organisms where initiation of an error catastrophe would have disastrous consequences. In higher organisms, however, the selective balance would be different, because the great majority of cells are somatic cells, and in these it does not matter so critically if a proportion of them degenerate to error catastrophe.
C . EVOLUTIONOF AGING Aging may be defined most conveniently as a process which renders individuals more susceptible as they grow older to the various factors which may cause death (Medawar, 1946; Maynard Smith, 1962; Kirkwood, 1984a). Although there are limitations to this definition based on survivorship (see Kirkwood, 1984a, for review), it is commonly recognized that it adequately describes the generalized deterioration which occurs in higher animals after they have passed their reproductive prime. Although it is not uncommon to find the term aging applied widely to plants, invertebrates, and certain microorganisms, there are major difficulties in adapting a single term to describe meaningfully an extreme diversity of life-history patterns (Kirkwood and Cremer, 1982; Kirkwood, 1984a). In this section we consider only the aging of higher organisms which undergo repeated sexual reproduction during their lifetime, and we ex-
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clude therefore all organisms with asexual life-histories, as well as organisms, such as Pacific salmon, which reproduce only once and then die in a determinate fashion. The aging of an organism which reproduces repeatedly has long been recognized as an evolutionary puzzle, since it would clearly be more advantageous to the individual to survive and reproduce indefinitely (Medawar, 1952; Williams, 1957; Hamilton, 1966; Kirkwood, 1977, 1981; Kirkwood and Holliday, 1979). The theory that aging is an evolved mechanism to promote species’ adaptability rests on the weak principle of group selection (Maynard Smith, 1976), and is not generally tenable. Therefore, it is recognized that evolution of aging is explained either as arising through the failure of natural selection to prevent it (see Medawar, 1952) or as a by-product of selection for some other trait (see Williams, 1957; Kirkwood and Cremer, 1982). The disposable soma theory of aging (Kirkwood, 1977, 1981; Kirkwood and Holliday, 1979) provides evolutionary support for the idea that aging is due to an energy-saving strategy of investing only the minimum necessary amount of resources in the maintenance of somatic cells. This in turn provides support for the hypothesis, suggested by Orgel (1963, 1973; see also Medvedev, 1962), that aging is due to instability in the cellular translation process. The starting point for the disposable soma theory is the observation, made orginally by Weismann (see Kirkwood and Cremer, 1982), that in multicellular organisms cells may be classed into two primary groups, reproductive cells and somatic cells. Reproductive cells constitute essential links in the chain of inheritance of genetic information and need a high degree of translational stability, since progressive deterioration in fidelity would be disastrous. By contrast, among somatic cells the future lineage of any single cell is limited since it is certain that, even in the absence of aging, the body of which it forms a part will eventually die. In somatic cells there is therefore not the same need for long-term stability, and this leaves the organism freer to vary its investment in translational accuracy and stability so as to maximize its lifetime reproductive success, or fitness. A trade-off which is of particular significance here is between the organism’s investment in reproductive output and its investment in translational stability. The larger the fraction of resources invested in translational stability, the smaller the investment in reproduction (and conversely), irrespective of whether resources are abundant or scarce. At either extreme, the organism pays a penalty. If investment in translational stability is too little, cells will become rapidly destabilized and death will result very soon. If investment in translational stability is too much, reproduction will be severely retarded. In consequence, the strategy of maximum fitness is one which optimally balances survival against reproduction, and it may be shown that the ideal investment of resources in mechanisms to promote survival is likely to be less than would be required for the organism to survive indefinitely (Kirkwood, 1981). To the extent that sur-
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viva1 is dependent on the preservation of translational stability, the disposable soma theory predicts therefore that aging may result from an increasing proportion of somatic cells becoming destabilized. The theory also predicts that longer lived species have, in general, a higher level of translational stability than shorter-lived species (Kirkwood and Holliday , 1979).
IV. Nature of Proof or Disproof Over the 20 years since the error catastrophe hypothesis was put forward, it has become increasingly clear that it is an extremely difficult concept to test. There are two main reasons for this. First, the error level in translation is so low that it is hard to measure it sufficiently accurately to be confident of detecting a change (see following section). Second, it is unclear how great an increase in error frequency would be necessary to produce an error catastrophe; it is possible that even quite a small increase could have a profound effect on cell viability. The term “error catastrophe” is probably quite misleading in that it suggests a state of presumably obvious disintegration. In fact, a relatively modest increase in error level from, say, to l o p 3 mistakes per amino acid residue would reduce the proportion of error-free protein from 99 to 90% for proteins of 100 residues, and from 90 to 37% for large proteins of 1000 residues. Such a change, together with a corresponding increase in the cell’s rate of protein turnover as scavenging mechanisms try to keep pace with new errors, would substantially reduce a cell’s metabolic efficiency and may be sufficient to prevent it from undergoing further cell division. At the same time, an increased level of faulty enzymes would render more likely one of the serious faults which kills a cell or blocks it from cycling. Despite these difficulties, numerous attempts have been made to measure the accuracy of the translation process and to determine the extent of error propagation in present-day cells (for earlier reviews, see Rothstein, 1977; Gershon, 1979; Medvedev, 1980; Gallant, 1981; Laughrea, 1982). These studies have followed two principal lines. In the first, which relates directly to the error theory of aging, cells such as mammalian fibroblasts which routinely undergo clonal extinction have been examined for evidence that the error frequency increases during clonal growth. In the second, cells such as bacteria, which would be regarded as normally stable, have been subjected to treatments known to increase the frequency of mistranslation in attempts to induce translational instability. That the results have been interpreted in conflicting and occasionally irreconcilable ways is due to the fact that the predictions which have been tested have tended to be indirect and they have not always been sufficiently clearly formulated. The most direct form of experimental test would be to measure the rates at
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which various cell types make errors and to follow how these rates changed during clonal growth, or after sudden perturbations of fidelity. If it could be established unequivocally that the error rates do not progressively change, then the hypothesis of translational instability would be effectively disproven. On the other hand, if the error rate were found to increase, this would strongly support the error propagation hypothesis; final proof would require demonstration that active, erroneous information handling molecules were being made and that other changes which might affect translational accuracy could effectively be ruled out. Current methods for measuring error rates are not without limitations, however, and various indirect tests are therefore widely used. For example, if error propagation does take place an increasing proportion of molecules of each specific type of protein will contain one or more amino acid substitutions, and these may be detectable through a lowering of the average specific activity of the protein, or an increase in its thermal instability. Alternatively, viruses have been used as probes of translational instability on the grounds that if translational stability has broken down, then an increasing proportion of defective virus particles should be produced. The drawback to any indirect test is, however, that a positive or negative result may be an artifact of the system, rather than a consequence of the truth or falsehood of the primary hypothesis. Gallant (1981) has stressed the logical fallacy of basing any firm conclusion on an experiment of the indirect type, where more than one explanation can usually be offered for any given observation. Nevertheless, indirect tests do have a role to play in adding to or subtracting from the total weight of evidence which bears on the validity of error catastrophe hypothesis. A special kind of indirect test involves comparative study of cells from different species or of different types. For instance, the disposable soma theory suggests that if translational instability is the primary cause of aging, then cells from long-lived species should be more stable than cells from short-lived species (Kirkwood and Holliday , 1979). Similarly, somatic cells from multicellular organisms are predicted to be less stable than unicellular organisms and this too should be amenable to test. Finally, transformed cells, grown either from a malignant tumor or obtained by treatment of normal cells with carcinogens, grow indefinitely in culture in contrast to their normal counterparts; this last difference is explicable in terms of the error theory if either selection against destabilized cells is stronger in more rapidly growing transformed cell populations because translational efficiency becomes rate-limiting for cell division (Holliday, 1975; Kirkwood and Holliday, 1975b), or reactivation occurs in transformed cells of special error-correcting mechanisms which normally operate only in germ cells (Kirkwood, 1977). It should be noted that each of these comparative predictions relates primarily to the stability of translation, whereas comparisons are most easily made of the accuracy of translation. The HKH model illustrates how
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FIG.6. The adaptor model of error propagation (see Section 11) indicates that accuracy and stability of translation are to some extent independent. Curves A and B have the same level of stable accuracy, qstable, but curve A has a lower threshold than curve B and is therefore more stable. Such a situation could arise if curve A represents a translation apparatus with lower values of R and S than curve B. (Only the upper right regions of the curves are shown.)
stability and accuracy, although related, are also partially decoupled (Fig. 6), and it is thus possible that accuracy might be more or less the same, while stability is quite different. To conduct a proper comparative analysis of translational stability it is necessary, therefore, to measure protein turnover rates (see Section VI) as well as the error rates in protein synthesis. Finally, tests can be made of models of error propagation as well as of the general predictions of the error catastrophe hyopthesis itself (see, for example, Gallant and Prothero, 1980). These tests have the advantage that models generally make quantitative predictions, so that tests can be more exact, but it is mistaken to regard a test of a model as necessarily the same thing as a test of the theory which the model represents. In fact, for reasons discussed earlier, none of the error propagation models to date can be regarded as sufficiently realistic to warrant detailed quantitative test. This does not mean, however, that the models have no role to play in experimental test of the error propagation hypothesis. For example, the HKH adaptor model provides a useful background to a series of experiments in Escherichia coli in which aminoglycoside antibiotics, which increase the rate of ribosomal mistranslation. were added to the medium in an
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FIG.7. Effect of streptomycin on the accuracy of translation in a stable organism (e.g., E . coli), according to the adaptor model (see Section 11). Without streptomycin, the accuracy remains at the stable point A on the upper curve. Adding streptomycin decreases specificity, S, and displaces the curve DA down to the curve CB. Accuracy falls to the lower stable point, C. On removing streptomycin, curve CB is restored to DA and accuracy returns to the stable point A. In practice, there would be time lags while streptomycin is taken up by the cells and diluted out from them, so the changes in accuracy between A and C, and back again, would not follow exactly the indicated pathways but would be more gradual. If curve CB had no stable point (not shown) accuracy would fall progressively after addition of streptomycin and would only return to A on removal of streptomycin if it had not already fallen below qthCerhold for curve DA. Note that the figure shows only the upper right regions of the curves. (From Kirkwood, 1980.)
attempt to induce error catastrophe (see Section VII). The effect of adding antibiotic to the culture medium is roughly equivalent to decreasing the specificity parameter, S, causing a downward displacement of the curve. Depending on the dose and cell strain used, this may or may not eliminate the stability of translation, but even if it does not, qstableis reduced (Fig. 7). On removing the antibiotic the system should return to the original qstableprovided accuracy has not already fallen below the normal qthreshold. In this way judicious experimentation can test the general stability characteristics of the model, as defined by the critical points shown in Fig. 7. However, the precise pathways taken between the points will depend both on the kinetics of uptake and clearance of antibiotic from
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the cell and on more complex realities of the translation process than are currently represented in the model.
V. Measurement of Error Rates A. PROTEINERRORS A number of factors combine to make the determination of protein errors a difficult and challenging problem. Mistakes in transcription or translation will introduce errors at random sites in the polypeptide chain and any residue may be replaced by several alternatives. Purification and detection are therefore much more complex than with isozymes or mutant proteins. In the latter two cases, proteins differ by unique substitutions and can often be resolved into distinct bands or peaks by chromatography or electrophoresis. No such bands will appear when the substitutions are random, and the likelihood of losses during purification is very great. Despite these difficulties, several approaches have been described which, potentially at least, could detect error rates as low as one mistake in 103-105 codons read. None of these as yet provides a complete solution to the problem, but all are capable of further refinement and they have provided the most incisive data available at present. The principles on which they are based and their attendant advantages or disadvantages will be discussed in this section. 1, Radiolabeling of Proteins with Specific Amino Acids
When the structural gene of a protein does not specify the insertion of a particular amino acid, it will appear in the polypeptide only through a decoding mistake. This leads to a relatively straightforward method of measuring errors. The normally missing amino acid is supplied in radioactive form during protein synthesis, the protein purified to homogeneity, and its radioactivity determined. The assays can be made very sensitive by using amino acids of high specific activity. This approach can further be modified for use on proteins which do contain all 20 amino acids. Most proteins can be hydrolyzed enzymatically into specific polypeptide fragments. If any fragment does not contain all 20 monomers it could be used to determine error frequencies after labeling with the missing amino acid (Loftfield and Vanderjagt, 1972; Bouadloun et al., 1983). While it has great potential, there are several difficulties inherent in this whole approach. The most serious problem is the need to purify the protein so that traces of other polypeptides, containing the labeled amino acid, do not interfere. Errors which change the properties of molecules so that they no longer copurify with the correctly assembled polypeptide will not be detected. Further, isolation
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procedures are relatively laborious and this limits the number of samples which can be processed. This is particularly true when the protein has to be degraded into peptides which themselves have to be purified. Finally, the choice of proteins is somewhat limited, for most polypeptides do contain all 20 amino acids and the larger proteins give rise to a forbidding array of proteolytic fragments. Radiolabeling has been used to study accuracy in the synthesis of a mammalian histone lacking methionine (Buchanan and Stevens, 1978; Medvedev and Medvedeva, 1978), of flagellin from E. coli which has no cysteine (Edelman and Gallant, 1977a), of the E. coli ribosomal protein L7/L12 lacking cysteine (Bouadloun et al., 1983) and of chymotryptic and tryptic peptides from rabbit hemoglobin and chick ovalbumin which do not contain leucine, isoleucine, or valine (Loftfield, 1963; Loftfield and Vanderjagt, 1972). The results obtained from these proteins are summarized in Table I and discussed in a later part of this section. 2. Miscoding for Nonsense and Frameshgt M ufations Mutations can alter certain amino acids to read as end of translation signals (nonsense codons). They can also, by the addition or deletion of bases, shift the reading frame to produce garbled messages. One feature of both these mutational changes, not necessarily shared by missense mutations, is that when they occur in the structural gene of an enzyme they lead to a completely inactive protein being formed. Active enzyme will then only be made after a decoding mistake, and enzyme assays can be used to measure error rates (Gorini, 1970; Gallant and Foley, 1980). With some enzymes, particularly hydrolases like P-galactosidase and alkaline phosphatase, activities as low as l o p 5 of the normal can be detected (Gallant and Palmer, 1979; Rosenberger et al., 1980). This approach has the advantages of requiring only small amounts of material and of being far simpler and more convenient than any other so far described. Its main drawback is that it does not measure the substitution of one amino acid for another but, rather, errors in discrimination between release factors and noncognate amino acids (nonsense mutations) or the rate at which irregular ribosome movements and transcriptional errors occur (frameshift mutations). These aspects of fidelity are not necessarily the same. Further clarification of the suitability of multimeric enzymes, such as P-galactosidase and alkaline phosphatase, for this assay is also needed. Each decoding mistake produces a single subunit which will become active only when combined with other subunits, themselves produced at rare and random intervals. Ideally, an enzyme should be used that is active as a monomer. To date, readthrough assays with nonsense and frameshift mutations have been limited to prokaryotes and lower eukaryotes, since it is only in these organisms that such mutations have been detected with certainty. Results are given in Table 1.
TABLE I ERRORRATESDURING PROTEIN SYNTHESIS B Y VIGOROUSLY GROWING OR YOUNGCELLS ~~
Assay method
Organism
Incorporation of normally missing amino acids
Man Human fibroblasts Mouse E . coli E. coli
Misincorporation at a specific codon Readthrough of nonsense mutations
Protein
AA measured
Hemoglobin Histone HI
Ile Met Met CYS
Rabbit E. coli E . coli
Histone HI Flagellin Ribosomal protein L7lL12 Hemoglobin MS2 coat protein P-Galactosidase
E. coli
Alkaline phosphatase
“Misassignmentslcodons read
CYS
Val for Ile Lys for Asn
-
Error rate“ 3 x 10-5