No title

Current Topics in Membranes and Transport VOLUME 29

Membrane Structure and Function

Advlsory Board

G . Blobel

E. Carafoli J . S. Cook D.Louvard

Current Topics in Membranes and Transport

VOLUME 29

Membrane Structure and Function

1987

ACADEMIC PRESS,INC. Harcourt Brace Jovanovich, Publishers

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Contents

Preface, ix Yale Membrane Transport Processes Volumes, xi

Current Views of Membrane Structure ALAN M. KLEINFELD 1. 11. 111. IV. V. VI.

Introduction, I Lipid Structure, 3 Lipid-Lipid Interactions: Single Lipid Species. 4 Lipid Mixtures and Domains, 10 Membrane Protein Structure. 12 Interactions between Lipids and Proteins, I6 References. 20

Ultrastructural Studies of the Molecular Assembly in Biomembranes: Diversity and Slmilarity SEK-WEN HUI 1. 11.

Ill. IV. V.

VI.

Similarity and Diversity of Biological Membranes. 30 Is the Lipid Bilayer a Passive, Homogeneous "Solvenl"'!. 35 Is the Bilayer the Only Form of Membrane Lipids?, 43 Do Membrane Proteins Distribute Randomly in the Bilayer'?, SO What Determines the Conformation of Intrinsic Protein Molecules in Membranes'?, 57 A Look at the Future through the Electron Microscope, 62 References. 65

vi

CONTENTS

The Thermodynamics of Cell Adhesion MICAH DEMBO AND GEORGE 1. BELL I. 11.

Ill. IV. V. V1. VII.

Introduction, 71 The Model, 71 The Chemical Potentials, 74 The Repulsive Potential, 76 Minimizing the Free Energy, 78 Results, 82 Discussion, 87 References, 89

Rotational and Lateral Diffusion of Membrane Proteins and Lipids: Phenomena and Function MICHAEL EDIDIN Introduction, 91 Probes for Diffusion, 92 111. Rotational Diffusion, 93 IV. Lateral Diffusion, 102 V. A Concluding Remark, I I9 References, I I9 1.

11.

Biosynthesis and Distribution of Lipids KENNETH J . LONGMUIR Introduction, 129 The Lipid Composition of Subcellular Membranes, 130 Desaturation and Elongation of Fatty Acids, 141 IV. Phospholipid Biosynthesis, 144 V. Membrane Lipid Tmnsport and Differentiation of Membranes, 163 References, 167 I.

11. 111.

Lipid Exchange: Transmembrane Movement, Spontaneous Movement, and Protein-Mediated Transfer of Lipids and Cholesterol ELlEZAR A. DAWIDOWICZ Introduction, 175 Spontaneous Exchange of Lipids between Membranes, 176 Protein-Mediated Lipid Transfer between Membranes, 186 IV. Transmembrane Movement of Lipids. 189 References. 193

1. 11. 111.

CONTENTS

Membrane Fusion ROBERT BLUM ENTH AL I. Introduction. 203 11. How We Observe Membrane Fusion. 205

I l l . 'The Triggering Event in Membrane Fusion. 213 1v. Movement into Apposition, 216 V. The Recognition Event, 219 VI. Steric Constraints. 22 I VII. Motion of Phospholipids, 223 VIII. Interactions between Bilayer Membranes, 227 I X . Membrane Fusion: Fact. Hypothesis, or Theory'?, 240 References. 245

The Control of Membrane Traffic on the Endocytic Pathway I K A MELLMAN, CHRISTINE HOWE. AND AKI HELENIUS 1. Introduction, 255 11. Endocytosis and Membrane Recycling, 2.56 111. Keceptor-Mediated Endocytosis. 258 IV. Mechanisms and Functions of Endocytic Organelles. 260 V. The Exocytic Pathway. 279

References. 281

Index, 289 Contents of Recent Volumes, 299

vii

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Preface The science of membrane structure and function has seen significant progress over the past 15 years. Needless to say, we are only beginning to understand the intricacies involved in maintaining the compartmentalization that is needed to restrict and regulate metabolic processes within the living cell and to permit an adequate, controlled dialogue with the extracellular environment. It has become increasingly clear that membranes themselves are highly dynamic structures, from a physical as well as a chemical point of view. The study of their structure and function requires sound hypotheses to work from as well as the application of a great variety of biophysical, biochemical, and morphologic techniques. Concepts derived from detailed investigations into t h e properties of artificial model membranes are being utilized more and more to gain fundamental insights into the physiology of biomembranes. Volume 29 of Ciirrerit Topics in Mrmhrtrnc~sand Transport is meant to highlight some of the paths in the large field of membrane biology along which recent progress has been realized and to delineate objectives toward which future investigations might be aimed. Many articles in this volunie carry elements of both a review and an essay in an effort to invite the reader to regard them as an introduction to current thinking rather than only a s a review. In the first article, a broad overview is given of our current knowledge of membrane structure in general. A number of morphological backups for these concepts as well as further, new observations on membrane ultrastructure are given in the second article. Communication between cells and between cells and their environment is mediated by membranes and membrane-based molecules. Theoretical models can be devised which may point to specific molecular mechanisms in such communication. The third article presents an example of building a model for cell adhesion in which density and mobility of membrane surface receptors appear as important parameters. The subsequent fourth article explores many theoretical and experimental aspects of the dynamics of proteins and lipids in the mcmbl-me. In the fifth and sixth articles, topology and metabolism of membrane lipids along with implications for the transmembrane distribution of lipids and the compositional diversity of cellular membranes are discussed. The study of fusion processes in artificial and biological membranes has been ix

X

PREFACE

greatly facilitated by the development, over the past 6 years, of a variety of assays. The seventh article provides a survey of membrane fusion studies, the hypotheses behind them, and the models arising from them. Recent, new understanding of the process of endocytosis and its underlying membrane traffic has given a boost to the investigation of such complicated matters as intracellular routing and sorting in which the precise role(s) of membranes and membrane constituents is still largely unknown. The final article describes the developments in this field and offers a number of leads to further experimental approaches. Virtually any volume on progress in the study of membrane structure and function is bound to be far from complete. This volume is no exception in this respect. As our knowledge of membrane physiology increases, essays and reviews on the subject will render an increasingly integrated picture. JOS V A N RENSWOUDE CHRISTOPH KEMPF RICHARD D. KLAUSNER

Yale Mem bra ne Transport Processes Volumes Joseph F. Hoffman (ed.). (1978). “Membrane Transport Processes,” Vol. I . Raven, New York. Daniel C . Tosteson, Y u . A. Ovchinnikov, and Ramon Latorre (eds.). (1978). “Membrane Transport Proccsses,” Vol. 2. Raven, New York. Charles F. Stevens and Richard W. Tsien (eds.). (1979). “Membrane Transport Processes,” Vol. 3 : Ion Permeation through Membrane Channels. Raven, New York. Emile L. Boulpaep (ed.). (1980). “Cellular Mechanisms of Renal Tubular Ion Transport”: Volume I3 of Crirrctil Topics in Mcr?ihmnes rind Trrinsport (F. Bronner and A. Kleinzeller, eds.). Academic Press, New York. William H. Miller (ed.). (198 I ) . “Molecular Mechanisms of Photoreceptor Transduction”: Volume 15 of Cirrrcnt Topics in MonbrLines mid Trruisport (F. Bronner and A. Kleinzeller, eds.). Academic Press, New York. Clifford L. Slayman (ed.). (1982). “Electrogenic Ion Pumps”: Volume 16 of C‘irrrrnt Topics in Mrmbrrinrs rind Trtinsport ( A . Kleinzeller and F. Bronner, eds.). Academic Press, New York. Joseph F. Hoffman and Bliss Forbush 111 (eds.). (1983). “Structure, Mechanism, and Function of the Na/K Pump”: Volume 19 of Crrrrcnt Topics in Mrnihrrincs cind Trrinsporf (F. Bronner and A. Kleinzeller, eds. ). Academic Press, New York. James B. Wade and Simon A. Lewis (eds.). (1984). “Molecular Approaches to Epithelial Transport”: Volume 20 of Crrrrenf topic..^ in Mombrrinc>s mid Trrrtisport (A. Kleinzeller and F. Bronner, eds.). Academic Prcss, New York. Edward A. Adelberg and Carolyn W. Slayman (eds.). (198s). “Genes and Membranes: Transport Proteins and Receptors”: Volume 23 of Crrrrcnt Topics in Memhrtines und Trrrnsport (F. Bronner and A. Kleinzeller, eds.). Academic Press, Orlando. Peter S . Aronson and Walter F. Boron (eds.). (1986). ‘“a’- H’ Exchange, Intracellular pH, and Cell Function”: Volume 26 of Cirrrenf Topics in Mcmhrcrnc~sand Trrrnsport ( A . Kleinzeller and F. Bronner, eds.). Academic Press, Orlando. Gerhard Giebisch (ed.). (1987). “Potassium Transport: Physiology and Pathophysiology”: Volume 28 of Cirrrent Topics in Mctnhrrrncs tind 7’rrrnsport (F. Bronner and A. Kleinzeller, eds. 1. Academic Press, Orlando.

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CLIRRENT 1 0 P I C S IN MEMBRANES AND TRANSPORT. VOLLIME 29

Current Views of Membrane Structure ALAN

M. KLEINFELD

1.

INTRODUCTION

The current view of membrane structure is similar in many respects to the general outline of membrane structure developed more than a decade ago by Singer and Nicolson (1972). The essential features of the model, consisting of transmembrane and peripheral proteins in association with a lipid bilayer, remain intact. In the intervening decade a more profound understanding has been achieved for lipid and protein structure, the interactions between these membrane constituents, and the dynamics of these interactions. This article presents an overview of recent developments in the structure of membrane components and certain of their interactions. Subsequent articles will further explore the structural and dynamic aspects of these interactions. The rationale for studying the structure of membrane components is that such knowledge will allow one to predict the form of the composite membrane and ultimately to understand membrane function at the molecular level. Current understanding of the constituent structures and their interactions leaves us far from this goal. Where the relationships between molecular structure and function are well understood, as for example in the cases of lysozyme (Phillips, 1967) and hemoglobin (Perutz et d., I%%), physiological function appears to be associated with structural changes that are on the order of I A, suggesting that this resolution is necessary to predict and understand function. Furthermore, because of the dynamic nature of the components forming the membrane, appreciable temporal resolution is also necessary. Thus the molecular coordinates determined from lipid and protein crystals must be modulated by molecular dynamics 1 Copyrighi lO"/sec) about the C-I-C-2 axis and, of course, the molecule as a whole rotates about an axis normal to the surface. According to the crystal structure, a polar region can be defined which extends from the head group to the carbonyls of the glycerol backbone and has a thickness, depending upon head group and acyl chain length, of between 8 and 10 These results are in excellent agreement with the electron density profile obtained by X-ray diffraction on aqueous dispersions (Blaurock. 1982). Thus a significant fraction of the bilayer thickness may form a region of relatively high dielectric constant [also suggested by electron spin resonance (ESR) and fluorescence studies, see below]. The acyl chain orientation is parallcl to the surface normal in the liquid crystalline state and is either parallel or tilted to the normal in the gel state. In either case, however, the phospholipid crystal structure (Hauser et a / . . 1981) as well as N M R studies of model and biological membranes (Seelig and Seelig, 1980) indicate that the first two carbons of the P-chain

A.

4

ALAN M. KLEINFELD

Y-chain h

a-ch

B-chain FIG.I . X-Ray crystallographic structure of phosphatidylcholine. A similar structure was obtained for phosphatidylethanolamine.(Reprinted with permission from Hauser ct a/.. 1981.)

are parallel to the surface and therefore the P-chain does not extend as far into the bilayer as the y-chain. An important consequence of these studies is that structures determined from the solid crystal can be applied to the fully hydrated state. Even the rotational mobility of the head group about the C-14-2 axis can be inferred from the single crystal structures. On the other hand the strong hydrogen bonding between head groups which is observed in the crystal study is probably not as significant in the fully hydrated state, at least at temperatures above the phase transition where the average separation of head groups exceeds hydrogen bonding distances. Thus while the crystal structure provides an essential starting point for using constituent structures to predict the form of the physiologically interesting aggregate, information from other sources will be necessary to completely understand membrane structure. 111.

LIPID-LIPID INTERACTIONS: SINGLE LIPID SPECIES

A. Bilayer Structure

Aqueous dispersions of phospholipid give rise to a number of distinct structures (Luzzati, 1968) including the bilayer, hexagonal tubes, and micelles, as shown in Fig. 2. The nature of the phase depends on lipid species, lipid concentration, temperature, pH, and ionic strength. Attention has focused on the nonbilayer or hexagonal tube phase which has been found to coexist with the bilayer phase for special lipid mixtures in model systems (Cullis and de Kruijff, 1979; Hui ef al., 1981). Since there is little evidence that nonbilayer and bilayer structures coexist in biological membranes the following section will deal primarily with the bilayer phase.

CURRENT VIEWS OF MEMBRANE STRUCTURE

5 C

Molecular Shape Lysophosphdipids Detergents

----

Micellar

-v-

- - .- _ _ .

Inverted Cone

Sphingomyelin ..

Bilayer

'hosphotldytethandamine (unsaturated)

.-

.

..

Cylindrical

---&

- - -

.. .

'hosphatidic acid Hexagonal (H,,:

. .. .

Cone

FIG.2. Phospholipid phases in aqueous dispersions. The structure of each phase is shown in H, examples of phospholipids which form such phases are given in A. and the overall shape o f the phospholipid molecule is shown in C. (Reprinted with permission from Cullis and de Kruijff, 1979.)

Specific conditions are required for bilayer formation. Phosphatidylcholines with acyl chain lengths greater than 12 carbons, for example, are capable of forming bilayer phases over a wide range of conditions which include the ones relevant to the physiological state. Bilayers of pure phosphatidylethanolamine, however, form only within a restricted range of temperatures and at pH greater than 1 I . Structural studies on single lipid crystals provide insight into conditions necessary for bilayer formation (Hauser et al., 1981). These results indicate that the phosphatidylethanolamine head group occupies an area of about 38 A? in both the anhydrous state and in the presence of excess water. The phosphatidylcholine head group. on the other hand, occupies an area of about 50 A' in the anhydrous state and about 60-70 A in the presence of excess water. Below the transition temperature the sum of the acyl chain cross-sectional areas (for

6

ALAN M. KLEINFELD

saturated chains) is about 20 A’, small enough for these chains to pack either perpendicular or tilted to the surface of both phosphatidylethanolamine and phosphatidylcholine bilayers. As the temperature is raised above the transition temperature the acyl chain area expands to 50 A2. The acyl chains will therefore curve outward from the head group, forming the inverted micelle shown in Fig. 2. For phosphatidylethanolamine, where head groups maintain tight packing even in excess water, acyl chain-head group packing constraints are not consistent with a bilayer phase. The micelles that are formed under these conditions are not stable in isolation and combine to form the hexagonal tube phase (Fig. 2). At elevated pH, phosphatidylethanolamine acquires a net charge and the resulting electrostatic repulsion creates a sufficiently large head group area to accommodate a bilayer phase. In contrast, the head groups of hydrated phosphatidylcholine occupy a sufficiently large area for acyl chain expansion to occur within a bilayer phase. In the liquid crystalline state, in fact, the phosphatidylcholine head groups occupy a considerably greater area than is necessary to accomodate the acyl chains. Over a wide range of conditions, therefore, phosphatidylcholine is able to form bilayers. X-Ray diffraction studies on dispersions of phosphatidylcholines demonstrate that the thickness of the glycerohydrocarbon region increases from about 26 A for 12-carbon acyl chains to about 30 for the 18-carbon lipid (Cornell and Separovic, 1983). Since this increase is less than expected for fully extended acyl chains and since for the same variation in acyl chain length, the area per phospholipid is found to increase from 60 to 75 appreciable folding is suggested for the central portion of the acyl chains. The head to head width of biological membranes is between 40 and 45 A, according to X-ray diffraction studies on multilayer preparations (Blaurock, 1982). This value is consistent with the 30 A width of the glycerohydrocarbon region of an 18-carbon lipid and a head group to glycerol backbone thickness of between 5 and 7 A, from the lipid single crystal results (Hauser r f mf., 1981). The studies of the phosphatidylcholine bilayer thickness indicate that the terminal ends of the acyl chains fold rather than intercalate between the acyl chains of the opposite bilayer leaflet. This would seem to suggest that lipid-mediated coupling of the two bilayer leaflets (a possible transbilayer signaling mechanism) is not a significant factor in phosphatidylcholine bilayers. X-Ray diffraction and electron microscopy of phosphatidylcholines with mixed 10- and 18-carbon acyl chains suggest, however, that interdigitation, and therefore possible bilayer coupling, occurs when the bilayer is in the gel state (Mclntosh el nl., 1984; Hui ef nl., I 984).

A

A.

CURRENT VIEWS OF MEMBRANE STRUCTURE

7

B. Properties of the Bilayer Normal to the Surface The interior of the membrane is not well approximated by a uniform isotropic hydrocarbon fluid, as witnessed by several important properties of the bilayer that exhibit significant variation in the direction normal to the surface. The degree of order as a function of bilayer depth has been studied by 'H or "F NMK using lipids selectively labeled along the acyl chain (Seelig and Seelig, 1980; MacDonald e t a / . , 1984). The reflects the average orientation of each molecular order parameter S,,,,,, segment along the acyl chain. having a value of zero for the completely disordered isotropic chain and a value of unity for a chain in the alltrans state. Seelig and his co-workers have found that, except for a slight dip at the 2 position, Smo,is relatively constant between carbons I and 9 and then falls rapidly for positions deeper in the bilayer, reaching a minimum at the center. The value at the minimum is about 3-fold smaller than the maximum value at the surface of the bilayer. Similar profiles were obtained in the "F studies of MacDonald et a / . (1984). In the liquid crystalline state the profiles are relatively unaffected by fatty acyl chain substitutions. For Acho/c.p/nsma lcridlrrivii B membranes in the gel state. however, enrichment in palmitate. but not unsaturated fatty acids, abolishes the order profile gradient (MacDonald er a / . . 1984). The behavior observed in the 'H and '"F studies is characteristic of model and biological membranes. The order parameter profile has also been investigated by measuring the fluorescence decay anisotropy of the 11-(9-anthroyloxy)fatty acids (Vincent cr d . , 1982; Kutchai et d . , 1983; Storch and Schacter, 1985; A . M . Kleinfeld, unpublished observations). These fatty acids labeled at one of eight different positions along the chain are added exogenously to the membranes and can therefore be used to study a wide variety of membranes. In Fig. 3 the order parameter profiles determined by fluorescence anisotropy are shown for red cell ghosts, liposomes, and paraffin oil. These profiles are in good agreement with those obtained by NMR. The larger S values for ghosts presumably reflect the high cholesterol content of these membranes while, as expected, the isotropic solvent paraffin oil exhibits an almost flat profile. The order parameter reflects the ensembled averaged orientation and may not be simply related to the rotational rate or segmental mobility. Information on the acyl chain mobility has been obtained from "C-NMR and fluorescence decay anisotropy measurements (Lee rt N / . , 1974; Vincent ct a / . , 1982; Kutchai et a/., 1983: A . M . Kleinfeld, unpublished observations). The results of these studies indicate that the mobility profile does, in fact. parallel the order parameter variation.

ALAN

8

0

K

W

!

.42

-

v V

%a ~

M. KLEINFELD

.20

-

w

P I 0

0

+ +

3.6

v

V

t

t

0

v

t

v

t

V

4Y

I

I

I

J

7.2

10.8

14.4

10

ACYL CHAIN

POSITION

PIG. 3. Order parameter variation along fatty acyl chains a s determined by the decay anisotropy of the anthroyloxy fatty acids. Fluorescence decay anisotropies of the n-(9-anthroyloxy) fatty acids ( n between 2 and 16) were measured in red cell ghosts (U). small unilamellar vesicles of egg phosphatidylcholine (A). and parafin oil ( 1. Differential polarized phase lifetimes were measured at an excitation wavelength of 383 nm, and the results were analyzed according to Kutchai et N I . (1983). The order parameter was calculated as S = (r,/r,J"', where r , is the measured value at 383 nm and r, = 0.3. (From A. M. Kleinfeld. unpublished observations.)

+

The polarity of the bilayer displays appreciable variation with position along the acyl chain and probably reflects the degree of water penetration into the bilayer. The first measurements of the polarity profile were performed using the n-doxylstearic spin-labeled fatty acids (Griffith et al., 1974). These electron spin resonance (ESR) measurements, in fully hydrated membranes, demonstrated that the polarity at the C-5 position was greater than ethanol while the polarity between the C-12 and C-16 positions was intermediate between mineral oil and ethanol. In dehydrated membranes, however, all probes sensed a similar polarity, suggesting that the observed gradient was due to water penetration. Confirmation of the hydrated membrane profile has been obtained by fluorescence lifetime studies of the n-(9-anthroyloxy) probes (Chalpin and Kleinfeld, 1983). The degree of water penetration appears to be modulated by lipid composition (Simon et al., 1982). In membranes composed of 1 : I phosphatidylethanolamine: cholesterol the depth to which a significant amount of water can penetrate

CURRENT VIEWS OF MEMBRANE STRUCTURE

9

appears to decrease by about 3.5 A . compared to pure phosphatidylet hanolamine.

C. Phase Transitions

Phase transitions in lipid bilayers have provided an important tool for the investigation of membrane structure. The phenomenon is a collective one and is thought to hold the key to many important cellular events. It is readily observed in model bilayers and its occurrence (in the form of lateral phase separation, discussed below) has been observed in some cellular membranes (Chapman. 197s; Nagel, 1980). Lipid bilayer phase transitions involve changes primarily in the acyl chains from a less to a more disordered state. There are a number of bilayer phases through which the transitions can proceed, and the occurrence of these phases depends primarily on the head group. Using the nomenclature of Ranck r t trl. (1974). the phases below the main phase transition are designated with a p and those above with an a. The main transition is accompanied by a significant enthalphy change (-9 kcal/mol) and is therefore considered to be first order although, since it occurs with a tinite width, it is not rigorously a phase transition (Nagel, 1980). In phosphatidylcholine with saturated acyl chains there is a state in which the chains are all trans, tilted to the bilayer, and in a monoclinic form; this state is designated L,.. In addition. there is another state (the P,. slate) in which the chains are also all trans and tilted but in addition a long range ripple is superimposed upon the monoclinic gel state. This state is more disordered than the L,. and therefore occurs at higher temperature (and is sometimes called the pretransition state). It has been observed (Chen et a / . , 1980; Ruocco and Shipley, 1982; Fuldner. 1981) that in dipalmitoylphosphatidylcholine the gel state is not an equilibrium state and that after prolonged incubation at 0°C the gel state slowly (>3.5 days) transforms into a more ordered state. Upon heating, the transition from this state (crystalline Lp,) to L,. is accompanied by a net enthalpy change. Thus a full description of the transitions in dipalmitoylphosphatidylcholine is L,$.(crystalline) -+ L,. (gel) P,,. + L,,. Only the main transition is observed in phosphatidylethanolamine, and this occurs at approximately 20°C above the corresponding phosphatidylcholine transition (Nagel, 1980). In addition to changes in the acyl chain order, the bilayer width decreases and the area per lipid increases for ;I L,, to L, transition. The increase in lipid area is also associated with an increase in hydration from 1 I to 15 molecules water per dipalmitoylphosphatid ylcholine below the main transition, to about 27 above.

-

10

ALAN M. KLEINFELD

IV. LIPID MIXTURES AND DOMAINS It was implicit in the original formulation of the fluid mosaic model (Singer and Nicolson, 1972) that the membrane constituents formed a wellmixed fluid. Numerous studies now indicate that membrane components exhibit varying degrees of immiscibility. Rates of protein diffusion (as discussed in Edidin, this volume) are generally inconsistent with movement restricted solely by a homogeneous lipid viscosity and in some instances may be consistent with zero mobility. Studies on the lipid composition of biological membranes generally demonstrate compositional asymmetry between the inner and outer bilayer leaflets and therefore an immiscibility of the lipids across the bilayer (see Dawidowicz, this volume). Our focus here is on lateral immiscibility of lipids. Lateral immiscibility in the form of lateral phase separation has been amply demonstrated in model systems (Shimshick and McConnell, 1973; Grant et al., 1974; Luna and McConnell, 1977). Phase separation has also been demonstrated in specialized biological membranes or those perturbed by enrichment or depletion of a particular component (fatty acid supplementation or sterol depletion). A more difficult issue, and one which has received considerable attention recently, is whether lipid immiscibility is a general phenomenon in biological membranes, resulting in small and consequently difficult to detect regions of lipid phase separation. Since these issues have been the subject of reviews elsewhere only a brief discussion of this subject will be presented here (Bach et al., 1977; Jain and White, 1978; Karnovsky et al., 1982a,b; Klausner and Kleinfeld, 1984). A. Model Membranes

Phase behavior has been studied in model membranes composed primarily of binary mixtures. Investigations have focused on mixtures of phosphatidylcholine, using virtually every biophysical method as well as many biochemical techniques. To a lesser degree phosphatidylethanolamine, sterols, sphingolipids, and charged phospholipids have also been studied, It is generally accepted that there are four different phases or phase mixtures in binary systems. (1) For lipids with nearly identical structure and/or phase behavior, bilayers exhibit nearly ideal mixing, so that above some critical temperature the composite system is entirely fluid, while below this temperature the acyl chains are all trans. This is virtually indistinguishable from a single species phase transition discussed above. True ideal mixing, therefore, implies near homogeneity in composition. (2) Binary mixtures of phosphatidylcholine, such as dimyristoylcholinedipalmitoylphosphatidylcholine and phosphatidylcholine-sphingomylin

CURRENT VIEWS OF MEMBRANE STRUCTURE

11

(Lentz rt d., 1976a,b, 1981), in which the components are similar, exhibit nearly ideal mixing but differ somewhat in their phase transition temperature. Thus above a critical temperature the lipids form an ideally mixed liquid phase. As the temperature is reduced, a value is reached where the presence of the higher melting point species causes solid domains to form in equilibrium with the fluid state lipid. The solid domains thus formed are not homogeneous in composition, but have a greater proportion of the higher melting component at higher temperatures. When the temperature is lowered sufficiently the entire phase is a well-mixed solid phase in which both components cocrystallize. (3) For lipids sufficiently different from one another the liquid phase will approach ideality. Lowering the temperature yields a coexisting fluid-solid phase. Finally, as the temperature is reduced further two solid phases will form in which the lower melting point component crystallizes separately from a phase composed primarily of the higher melting point component. (4) In a few special instances liquid-liquid immiscibility is exhibited. This behavior appears to depend on the nature of the lipids (Wu and McConnell, 1975; Galla and Sackmann, 1975) or the radius of curvature of the bilayer (Lentz et ( i l . , I976a,b). The nature of the liquid-solid phase separation has been investigated by combining freeze-fracture electron microscopy, electron diffraction, and ESR (Shimshick and McConnell, 1973; Grant pt ol., 1974). In all cases examined thus far phase separation appears to occur laterally within the plane of a single hemileaflet. Thus the separation does not correspond to separation into distinct membranes (fission), nor does it appear that the phases of the two leaflets of the bilayer are correlated. Lateral phase separations can be induced isothermally, as for example in the case of cation (Ca'+ ) applied to phosphatidylserine-phosphatidylcholine o r phosphatidylcholine-phosphatidylethanolamine mixtures. The detailed topology of the phase separation is difficult to assess although some evidence suggests patches with dimensions of the order of 0.2-0.5 Fm (Hui, 1981). 6. Biological Membranes

Biological membranes are obviously more complicated than the binary model membranes discussed above. The increased heterogeneity manifests itself both in head group and in acyl chain diversity; a considerable fraction of the acyl chains are cis-unsaturated and would therefore be expected to undergo phase transitions at very low temperatures compared to 37°C. In spite of these factors, membranes obtained from cells specifically enriched in a particular fatty acid or depleted in sterols, exhibit thermotropic behavior similar to binary model membranes (Melchior, 1982; Klausncr

12

ALAN M. KLEINFELD

and Kleinfeld, 1984). In addition, there are a number of cells which exhibit macroscopic lateral phase separation (Bearer and Friend, 1980; Wolf, 1983; Wolf and Voglmayr, 1984). These large-scale segregations of certain lipid classes are probably a result of lipid-lipid interactions as well as lipid interactions with other cellular constituents. In addition to these relatively large effects, there appears to be a class of phenomena, suggesting domains or clusters of lipids on a microscopic scale, for which the evidence is more indirect. The evidence for such domains has been obtained from calorimetry, photobleaching, and spectroscopy. Many of these effects have been discovered at temperatures appreciably below the physiologically important ones. Clearly a central issue is whether membrane lipid domains are significant structural factors at physiological temperatures. Several studies do in fact indicate domain formation under physiological conditions (Brasitus et al., 1980; Mutsch rt al., 1983). A number of cellular phenomena have been interpreted in terms of lipid domains. These phenomena generally involve the observation that altering fatty acid composition, either by changing the lipid acyl chain composition (Horwitz et al., 1974; Hatten rt al., 1978) or adding exogenous fatty acids (Karnovsky et al., 1982a,b; Hill et al., 1983; Klausner and Kleinfeld, 1984). alters a specific cellular function although the fatty acids remain unesterified. Modulation of cellular function by sterol alteration also suggests domains in lymphocytes (Hoover et al., 1983). These observations suggest a number of physiological roles which domains might play in cellular function. For example, depending upon their function, proteins may be located in regions where the lipids are in a solid phase and partitioning of lipophilic molecules (fatty acids, drugs, hormones) into this region may serve as a trigger for a particular function or as a larger region to collect a substrate for the protein. There are also a number of studies which suggest that domain formation itself may occur in response to physiological stimulus or growth (Curtain et al., 1980; de Latt et ml., 1979; Packard et al., 1984). V.

MEMBRANE PROTEIN STRUCTURE

Complete understanding of cellular function and the effects of lipids on this function must ultimately be based on the molecular structure of membrane proteins. Although atomic detail of membrane protein structure is not yet available, considerable advances have occurred in the past decade. A number of reviews have surveyed recent results (Senior, 1983; Benga and Holmes, 1984; Eisenberg, 1984). In this section the salient features of membrane protein structure will be reviewed, with emphasis on the

CURRENT VIEWS OF MEMBRANE STRUCTURE

13

portion of the protein associated with lipid. This latter qualifier is necessary since there are a number of membrane proteins that have large cxtramembranous domains which can be cleaved from the membrane-bound portion, crystallized, and studied by conventional X-ray diffraction (Mathews et ul., 1979: Wilson et ul., 1981). In contrast, the structure of proteins that are primarily lipid associated is at a much less refined level. Purification and crystallization for these proteins, for example, present much greater difficulties than for water-soluble proteins. The seminal study of membrane protein structure was performed on the photosynthetic reaction center using X-ray diffraction on the threedimensional crystals of this complex (Deisenhofer et ul., 1985). The naturally occurring two-dimensional crystals of bacteriorhodopsin formed in the purple membrane of Hdohacteriurn lirrlohirrm have been studied to partially solve the structure of this highly integral membrane protein (Henderson and Unwin, 1975). Using electron diffraction and microscopy an image of the protein was obtained at a resolution of 7 A parallel and 14 A perpendicular to the plane of the membrane. More recently this resolution has been improved to 6.5 and I!: A, respectively (Liefer and Henderson, 1983). Although at this resolution the location and orientation of individual amino acid residues cannot be discerned, a general outline of the protein shape and secondary structure can be inferred. The model of Fig. 4 indicates that the protein is composed of seven rods, roughly arranged as a cylinder of radius 15 A . Approximately 70% of the cylinder, whose length is about 40 is contained within the membrane. Based upon the analysis of the electron density, measurements of the circular dichroism (CD), the primary sequence, and theoretical arguments. it has generally been concluded that the seven rods are a-helicies and that the segments connecting the loops are extramembranous and nonhelical 1979; (Henderson and Unwin, 1975; Long r t 01.. 1977; Ovchinnikov et d., Khorana et ul., 1979; Engelman el ul., 1980; Senior, 1983). Recent diffraction studies and CD measurements suggest, however, that several of the rods may be partially @-sheet(Jap et id., 1983). Plausable models based upon the a-helical configuration have been proposed for the spatial arrangement of the primary sequence through the membrane (Engelman r t ul., 1982; Ovchinnikov et ul., 1979; Stoeckenius and Bogomolni, 1982). These models are consistent with the electron diffraction results and with labeling and proteolytic digestion studies which define the topology of the extramembranous regions (Engelman c’t rrl., 1982; Agard and Stroud, 1982; Huang et nl., 1982). Outlines of the membrane-associated portions of several other proteins have been obtained at lower resolution. These include gap junctions (Makowski et a / . , 1984; Unwin and Zampighi, I980), ~ibiquinol:cytochrome c reductase (Leonard et ul.. 1981), cytochrome oxidase (Deatherage rt

A,

14

ALAN M. KLEINFELD *.Q

@,",

.L.

11.

@'" "' "'

ALA C L I

**O@,mo

1.1.

*LA

@

@

ALA

PA)cl

scn 1*1-1.0

GLl

l C1

Leu-

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OL1

PLI LCU

.f*

ILC LCU

VAL

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100

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FIG.4. A model for the structure of bacteriorhodopsin. This proposed arrangement of the primary sequence through seven &-helices is based on electron diffraction, energy min-

imization. and biochemical evidence. (Reprinted with permission from Engelman 1982.)

ei

d..

al., 1982). and the acetylcholine receptor (Changeux et al., 1984). These

studies are generally consistent with a cylindrical shape for the membranespanning portion of the protein. In addition, high-resolution studies have been conducted on small membrane-associated peptides such as alamethicin (Fox and Richards, 1982) and melittin (Terwilliger et d., 1982). These studies have solved the structures at resolutions between 1.5 and 2.5 A,sufficient to determine the location of the amino acid side chains. The results are important for indicating how side chains interact with lipids and should be useful for analyzing larger proteins. In particular, the study on melittin suggests that hydrophobic residues associate with the lipid and hydrophilic residues orient toward the aqueous phase. A wide variety of experimental methods have been used to determine the location of particular portions of membrane-bound proteins and to relate these locations to the membrane architecture. These approaches complement diffraction methods that are as yet unable to distinguish individual residues. They also offer the possibility of monitoring at least

CURRENT VIEWS OF MEMBRANE STRUCTURE

15

part of the protein structure irr sitrr and possibly in different physiological states. Thus chemical labeling and proteolysis with selected proteases have been used to determine the number of times the protein loops across the membrane as well as the location and general topology of the extramembranous segments (band 3, Brock ct t i / . , 1983; bacteriorhodopsin, Engleman et a / . , 1980; histocompatibility antigen, Cardoza cr t i / . , 1984). Regions within the bilayer have been mapped out using feiritin labeling (Henderson et ( I / . , 19781, photoactivatable reagents attached to fatty acids (Robson cf a/., 1982; Takagaki et t i / . , 1983), and fluorescence quenching of tryptophan with spin-labeled fatty acids (London and Feigenson, 198 1a.b). Resonance energy transfer between tryptophan and lipid-associated acceptors has been used successfully to locate individual tryptophan residues and may allow the determination of the distribution of multiple tryptophan residues (Fleming ef d . , 1979; Kleinfeld and Lukacovic, 198.5; Kleinfeld, 198.5). Secondary structure of a number of membrane proteins has been studied by circular dichroism. Many of these studies suggest that the major portion 1977; of the hydrophobic region is a-helical (Guidotti, 1977; Long or d., Senior, 1983; Wallace ef id., 1986). Theoretical arguments have been presented suggesting that the a-helix is the energetically preferred configuration (Engelman and Stietz, 1981 ; Senior, 1983). Several circular dichroism and X-ray diffraction studies. however, have found appreciable p structure (Makowski e t a / . , 1982; Jap c t t i / . , 1983; Wallace er (11.. 1986). In fact. predictions of secondary structure based on the primary structure usually favor p-strand in the hydrophobic stretches of membrane proteins (Senior, 1983). p-Turns or -bends within the membrane have been thought to be energetically unfavorable, yet evcn these structures have been found in at least two cases (Dailey and Strittmatter, 1981; Cordoza ct a l . , 1984). Thus it is likely that the internal structure of membrane proteins is composed of both a-helix and p-sheet (Wallace ct t i l . , 1986). Since membrane protein structural information is so limited it is important to make use of theoretical as well as experimental information a s a guide in further analysis. Amino acid composition and primary structures have been examined in the hope that systematic behavior would emerge that would make it possible to characterize a protein as membrane or water soluble. The average hydrophobicity of the total amino acid composition does not appear to be a reliable predictor. Membrane proteins, however, i n contrast to water-soluble proteins, frequently possess stretches of hydrophobic residues that are long enough (>20 residues) to span the lipid bilayer. The correlation between hydrophobicity and the hydrophobic moment has been shown to be a reasonably successful prcdictor of protein classification (Eisenberg, 1984). On a plot ot of the hydrophobic moment versus the hydrophobicity per residue, proteins separate into surface (large moments and therefore asymmetric and relatively

16

ALAN M. KLEINFELD

small hydrophobicities), globular (intermediate moments and hydrophobicities), and transmembrane (small moments and large hydrophobicities). Hydrophobicity alone does not predict whether a residue will be located within the bilayer region since the most plausible models of bacteriorhodopsin, for example, suggest that a number of charged residues are located within the membrane (Engleman et al., 1980). To account for the considerable energy cost in burying a charged residue (20 kcal/mol) it has been suggested that the charged residues are distributed near the central axis of the cylinder formed by the seven rods and that hydrophobic residues are located at the periphery, in contact with the lipid hydrocarbon chains (Engelman and Zacci, 1980). VI. INTERACTIONS BETWEEN LIPIDS AND PROTEINS

Interactions between lipids and proteins have been studied by observing the effect of lipid on protein and the effect of protein on lipid structure. Proteins have been found to alter the order and mobility of the lipid acyl chain, the translational mobility of the whole lipid, and the lateral organization of the bulk lipid. The effect of lipid on protein structure and dynamics is less well characterized. A number of studies do, however, suggest alterations in specific protein segments in response to changes in lipid structure. In this section these two effects are discussed separately since they are studied by different methods and they potentially represent different physiological functions. A. Effects of Lipids on Proteins

Lipid-protein interactions involve the alteration in secondary, tertiary, or quaternary protein structure induced by changes in the composition or physical state of the lipid. Several studies in reconstituted systems using magnetic resonance have demonstrated that the mobility of individual amino acid side chains can be altered by changes in lipid phase (Hagan et al., 1978; Stollery et al., 1980; Boggs et ul., 1980). Secondary structural changes in response to alterations in lipid composition and lipid to protein ratio have been observed in reconstituted glycosyltransferase (Beadling and Rothfield, 1978) and fd coat protein (Dunker et al., 1982). Evidence also exists which demonstrates that fatty acid perturbation in plasma membranes may affect the conformation of individual amino acid residues (Esfahani and Devlin, 1982; Pjura et al., 1982). Interactions of small peptides with membrane indicate that binding occurs with a specific orientation relative to the membrane surface (Eisenberg, 1984) but that the actual

CURRENT VIEWS OF MEMBRANE STRUCTURE

17

conformational changes are quite subtle (Deber and Benham, 1984). A large body of indirect evidence for lipid-protein interactions has been obtained from functional changes observed in response to lipid and fatty acid alterations (Melchior and Steim, 1976; Criado et ul., 1984; Stubbs and Smith, 1984, Benga and Holmes, 1984). B. Effects of Proteins on Lipids

Protein-lipid interactions have attracted a great deal of attention in the past decade. This interest has been encouraged by the availability of a rich variety of lipid probes and by increased understanding of reconstitution of proteins into lipid bilayers. The central issue in these investigations has been whether and how the presence of protein in membranes affects lipid structure and dynamics. Proteins may affect the local properties of individual lipid molecules, such as the acyl chain order or mobility, or more global properties, such as the lateral and transmembrane distribution and mobility. Transmembrane effects will be discussed in Dembo and Bell (this volume) and lateral mobility in Edidin (this volume). In this section we will not deal with interactions affecting the transmembrane lipid properties and will only briefly touch upon lateral dynamics. The issue which has generated the most interest (and controversy) is whether a tightly bound lipid boundary is formed around transmembrane proteins. Tightness of binding is directly proportional to the residence time of a particular lipid in the protein-lipid interface. There are two important time scales with which to evaluate this residence time: the time characteristic of the protein’s function and the bulk lipid residence time. Since times which are important for protein function are probably greater than lo-‘ sec the lipid residence time should be greater than this in order for the binding to be functionally important. The bulk lipid residence time, the average time one lipid spends near another, is about lO-’sec (Marsh et al., 1982). This, therefore, is the shortest residence time in which any specific lipid can play a structural role. In this section we will pay particular attention to the issue of the lipid boundary, using these time limits to gauge the significance of protein-lipid interactions. The first direct physical evidence that lipid at the protein-lipid interface might exhibit special properties was obtained using cytochrome c oxidase reconstituted into liposomes composed of mitochondria1 lipid and a spinlabeled fatty acid (Jost et al.. 1973). The ESR spectra from such preparations suggest two environments, one in which the spin label exhibits the nearly isotropic tumbling that it does in pure lipid liposomes (only a single environment is detected in pure lipid) and one in which the probe is motionally restricted. Variation of the lipid to protein ratio demonstrated

18

ALAN M. KLEINFELD

that over a broad range of values the immobilized component was proportional to the protein concentration. At sufficiently high concentrations virtually all the probe was immobilized. From the observed saturation ratio it was estimated that the boundary lipid constituted one lipid layer or annulus around the protein. It was suggested that in normal membranes an annulus formed around integral membrane proteins and that the lipids in this annulus were tightly bound. Subsequent studies using ESR, fluorescence, and raman spectroscopy on several other reconstituted and plasma membrane preparations have confirmed the existence of immobilized lipid related to the presence of protein (Hesketh et al., 1976; Cable and Powell, 1980; Marsh and Watts, 1982; Boggs et al., 1982; Thomas et NI., 1982; Wolber and Hudson, 1982; Taraschi and Mendelsohn, 1980; Levin ec NI., 1982). The observation of an immobilized lipid fraction by these techniques does not, however, constitute evidence for immobilized lipid on the time scale of protein function or the bulk lipid exchange rate. These techniques have time windows which range from lo-* to sec. In the case of ESR, for example, it is only necessary for the spin label to exchange between the annulus and the bulk lipid at rates slower than about 108/sec for an immobilized component to appear in the ESR spectrum. NMR, which is sensitive to much slower events (exchange rates smaller than IO’lsec) can be used in the same way as ESR to detect regions of immobilized lipid. In most NMR studies no evidence of an immobile lipid component is observed, and therefore these studies establish a lower limit to the exchange rate of IO’/sec (Oldfield et al., 1978; Seelig et al., 1981, 1982). The constraints set by the ESR and NMR results therefore require that if bound lipid exists its mean residence time in the annulus is between IO-’and IO-’sec. Indeed, there is some evidence obtained from the analysis of the position and line shape of the ESR spectra that the fatty acid spin label exchange rate is about 107/sec(Marsh et al., 1982). This value is about the same as the bulk lipid exchange rate, suggesting that the exchange of lipid in the annulus with bulk lipid is unhindered and, therefore, that the annular lipid is not tightly bound to the protein. Spectroscopic techniques generally reveal a component which is rotationally immobile for times longer than the temporal resolution of the method and, therefore, only indirectly reflect lipid to protein binding affinities. Measurements of the effect of protein on lipid lateral mobility and the lipid-protein association constants represent more direct approaches to the question of how tightly lipid is bound to the protein surface. Several investigations using fluorescence photobleaching recovery to measure the lipid lateral mobility in the presence and absence of protein have been carried out in reconstituted and native membranes (Golan et al., 1984).

CURRENT VIEWS OF MEMBRANE STRUCTURE

19

Results in red cells indicate that the rate of lipid diffusion is slower by a factor of 4 in whole membranes a s compared to liposomes formed from extracted lipid (Golan el NI., 1984). This decrease, however, is entirely explicable in terms of lipid collisions with proteins acting as simple-hard surfaces, suggesting, therefore, that the protein-lipid interaction does not ex hi bi t a significant attractive interact ion. The association constant for the binding of lipid at the protein-lipid interface may be determined from the partition of lipid between the annulus and bulk regions, using spectroscopic markers to indicate the fraction in each region. In addition to the ESR method (Griffith c’t [ I / . , 1982). spinlabeled fatty acids and brominated phospholipids have been used as shortranged quenchers of intrinsic protein fluorescence, to determine the fraction of these probes in close apposition to the protein surface (London and Feigenson, 1981a,b; East and Lee, 1982). The results of these studies indicate that the partition of neutral lipid between the boundary and bulk phases is not significantly different from unity. Fluorescence energy transfer between tryptophan and fluorescent fatty acids suggests that the fatty acid is excluded from a region in immediate apposition to the protein. This is consistent with a preferential association of the phospholipid with the protein as compared to the fatty acid and with a tightly bound lipid annulus (Fleming ef ( I / . , 1979; Lee et ( I / . , 1982; Kleinfeld and Lukacovic, 198s). I t is possible, however, that this represents not tight binding of [he lipids to the protein surface but rather the decreased partition of the lipid probes into a region in which the acyl chain free energy is reduced by restrictions to rotation or by electrostatic repulsion between the protein and the charged probes. Although there is little evidence for specific binding of neutral lipid to the lipid-protein boundary, a number of studies suggest that tight binding may be exhibited by charged lipids and will, therefore, probably be protein specific. Proteins from myelin, studied by a number of techniques. appear to induce formation of domains, enriched in charged lipids, from mixtures 1982). The lipid in these domains of neutral and charged lipids (Roggs et d., as well as in the bulk phase retain their unmixed phase behavior suggesting that the segregation occurs without significantly affecting the acyl chain properties. Studies on cytochrome c’ oxidase and the matrix protein of vesicular stomatitis virus also indicate the segregation of charged lipids and demonstrate an approximately 2-fold increase in partition preference of the charged lipids for the boundary. in comparison to neutral lipid (Cable and Powell, 1980: Griffith ct NI., 1982; Wiener c’t ( I / . , 1983). The Ca” ATPase from sarcoplasniic reticulum, on the other hand, displays no binding specificity for charged lipids (East and Lee, 1982). Although proteins may not retard lateral diffusion of lipids other than

20

ALAN M. KLEINFELD

by providing simple barriers, a number of studies have demonstrated an effect on acyl chain order and mobility (the effect of proteins on transbilayer movement is dealt with in the article by Dawidowicz in this volume, where it is suggested that proteins tend to enhance rather than retard such movement). Deuterium-NMR studies in reconstituted systems of several proteins demonstrate that the degree of acyl chain order decreases in response to protein incorporation (Oldfield el al., 1978; Seelig et al., 1982). This may reflect the influence of the uneven, relatively convoluted surface of the protein on the packing of the neighboring lipid acyl chains (Seelig ef a!. , 1982). Measurements of the fluorescence decay anisotropy of the fatty acid analog paranaric acid confirm the decrease in order but also indicate that the rotational mobility of the lipid acyl chains is decreased by protein (Wolber and Hudson, 1982). The decrease in rotational mobility is consistent with the protein side chains acting to retard the gauche-trans rotation of the lipid methylene groups. Thus it appears that virtually all measured protein interactions are consistent with the protein acting to retard movement by virtue of the slow diffusion of the protein mass as compared to the lipid. These results can in fact be understood in terms of an “infinite” barrier presented by the protein. A lipid molecule adjacent to a protein differs from one in the bulk lipid phase since its possible jump positions are limited by the protein. As the relative protein concentration increases some of the lipids will also be trapped within aggregates of protein and display further immobility. Although these results suggest that on average there is no specific protein-neutral lipid interaction it should be emphasized that only a small fraction of membrane proteins has thus far been studied. ACKNOWLEDGMENTS

I would like to thank Dr. Judith Storch for informative discussions about membrane structure and for her wise counsel concerning the manuscript. I would also like to t h a n k Michael Toon and Sean Condon for their careful reading of the manuscript. This work was supported by a Grant-in-Aid from the American Heart Association and with funds contributed in part by the Massachusetts affiliate (83-789) and a grant from the National Science Foundation (PCM-830268). This work was done during the tenure of an Established Investigatorship of the American Heart Association and with funds contributed in part by the Massachusetts affiliate (82-174). REFERENCES Agard, D. A,, and Stroud, R. M. (1982). Linking regions between helices in bacteriorhodopsin revealed. Biophys. J . 37, 589-602. Bach, D., Bursuker, I., and Goldman, R. (1977). Differential scanning calorimetry and enzyme

CURRENT VIEWS OF MEMBRANE STRUCTURE

21

activity of rat liver microsomes in the presence and absence of I-tetrahydrocannabinol. Biocliim. Bio~phys.Acrci. 469, I7 1-1 79. Beadling. L.. and Rothfield. I,. I. (1978). Modulation of the conformation of a membrane glycosyltransferase by specific lipids. Proc. Nurl. Accid. Sc,i. U.S.A. 75, 3669-3672. Bearer, E. L.. and Freind, D. S . (1980). Anionic lipid domains: Correlation with functional topography in a mammalian cell membrane. Proc.. Ntrtl. Ac,tid. Sci. U . S . A . 77. 66016605. Benga, C . , and Holmes, R . P. (19841. Interactions between components in biological membranes and their implications for membrane function. I'rog. Biophvs. 43, 19.5-257. Blaurock. A . E . ( 1982). Evinence of bilayer structure and of membrane interactions from X-ray diffraction analysis. Bioc,/iirn. Biophy.~.Acrcr 650, 167-207. Boggs, J . M.. Stollery, J . G . , and Moscarello. M. A . (1980).Effect of lipid environment on (he motion of a spin-label covalently bound to myelin basic protein. Bio~hemi,sri:v19, 1226-1 234. Boggs. J . M., Moscarello. M . A,. and Papahadjopoulos. D. (1982). Structural organization of niyelin-Role of lipid-protein interactions determined in model systems. I n "LipidProtein Interactions" (P. C. Jost and 0. H. Griffith. eds.), Vol. 2 , p. I . Wiley. New York. Brasitus, T. A,. Tall, A. R.. and Schachter. I). ( 1980). Thermotropic transitions in rat intestinal plasma membranes studied by differential scanning calorimetry and fluorescence polarization. Bioc~lrc~rnistr?, 19. 1256-1261 . Brock, C. J . . Tanner. M. J. A,. and Kempf. C. (1983).The human erythrocyte anion-transpot1 protein. Bioc,hc.m. J. 213. 577-586. Cable. M. B . . and Powell. C. I,. ( 1980). Spin-labeled cardiolipin: Preferential segregation in the boundary layer of cytochrome c oxidase. Bioc~hrrni.sriy19, 5679-5686. Cardoza. J . I).. Kleinfeld, A. M.. Stallcup. K. C . . and Mescher, M. F. (19x4). Hairpin tr:~ configuration of H-2Kh in liposomes formed by detergent dialysis. B i o c ~ h c ~ ~ i r i s23, 440 1-4409. Chalpin. D. B . . and Kleinfeld. A . M. (1983). Interaction of fluorescent quenchers with the . 731, 4 6 5 4 7 4 . n-(9-anthroyloxy) fatty acid membrane probes. Bioc./iirn. B i o p l i y . ~Acfcr Changeux, J . , Devillers-Thiery, A,. and Cheniouilli, P. ( 1984). Acetylcholine receptor: an allosteric protein. ScYcnct, 225, 1335-1345. Chapman, D. (1975). Phase transitions and tluidity characteristics of lipids and cell membranes. Q . Rci.. Biophys. 8. 185-235. Chen. S . C.. Sturtevant, J . M . , and Gaffney. B. J . (1980). Scanning calorimetric evidence for a third phase transition in phosphatidylchoiine bilayers. Proc.. N i t / / .Ac,trif. S(.i. 1J.S.A. 77, 5060-SO63. Cornell. A , . and Separovic, F. (1983). Membrane thickness and acyl chain length. B i ( ~ ~ / i i i ~ i . Biopliys . A c'rtr 733, 189- 193. Criado. M., Eibl, H., and Barrantes. F. J . (1984). Functional properties o f t h e acetylcholine receptor incorporated in model lipid membranes. J . Biol. Chem. 259. 9188-9198. Cullis, P. R . . and de Kruijff. B. ( 1979). Lipid polymorphism and the functional roles of lipids in biological membranes. Biochim. Bioplrvs. Acrtr 559, 399420. Curtain. C . C.. Looney. F. D.. and Smelstorius. J . A. (1980). Lipid domain formation and ligand-induced lymphocyte membrane changes. Biocliirn. Biophps. Acrtr 5Y6, 43-56. Dailey. H. A , . and Strittmatter. P. (1978). Structural and functional properties o f t h e membrane-binding segment of cytochrome h,. J . Biol. Clrern. 253, 8203-8209. Dailey. H . A,. and Strittmatter. P. (1981). Orientation of the carboxyl and NH-. termini of the membrane-binding segment of cytochrome h, on the same side of phospholipid bilayers. J . Biol. C l r m . 256, 395 1-3955.

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Deatherage, J. F., Henderson, R., and Capaldi, R. A. (1982). Relationship between membrane and cytoplasmic domains in cytochrome c oxidase by electron microscopy in media of different density. J . M d . B i d . 158, 501-514. Deisenhoffer. J., Epp, O., Miki. K., Huber, R., and Michel. H. (1985). Structure of the protein subunits in the photosynthetic reaction centre of Rliodopsertdo/nonus viridis at 3A resolution. N o t ~ r e(London) 318, 618-624. Deber. C. M.. and Benham. B. A. (1984). Role of membrane lipids in peptide hormone function: Binding of enkephalins to micelles. Proc. N u t / . Acud. Sci. U . S . A . 81, 61-65, De Laat, S . W., Van Der Saag. P. T., Elson, E. L., and Schlessinger, J. (1979). Lateral diffusion of membrane lipids and proteins is increased specifically in neurites of differentiating neuroblastoma cells. Biockim. Biophys. Acta 558, 247-250. Dunker, A. K., Fodor, S . P. A,, and Williams, R. W. (1982). Lipid-dependent structural changes of an amphomorphic membrane protein. Biophys. J . 37, 201-203. East, J . M., and Lee, A. G. (1982). Lipid selectivity of the calcium and magnesium ion dependent adenosinetriphosphatase, studied d with fluorescence quenching by a brominated phospholipid. Biochemistry 21, 4144-4151. Eisenberg, D. (1984). Three-dimensional structure of membrane and surface proteins. Annrc. Rev. Biocliem. 53, 595-623. Engelman, D. M., and Steitz, T. A . (1981). The spontaneous insertion of proteins into and across membranes: The helical hairpin hypothesis. Cell 23, 41 1-422. Engelman, D. M., and Zacci, G. (1980). Bacteriorhodopsin is an inside-out protein. Proc. Nrrtl. A w d . Sci. U . S . A . 77, 5894-5898. Engelman. D. M., Henderson, R., McLachlan, A. D., and Wallace, B. A. (1980). Path of the polypeptide in bacteriorhodopsin. Proc. N u t / . Acud. Sci. U . S . A . 77, 2023-2027. Engelman. D. M., Goldman, A.. and Steitz, T. A. (1982). The identification of helical segments in the polypeptide chain of bacteriorhodopsin. Methods Enzyrnol. 88, 81-88. Esfahani, M., and Devlin, T. M . (1982). Effects of lipid fluidity on quenching characteristics of tryptophan fluorescence in yeast plasma membrane. J . B i d . C h e m . 257, 99199921.

Fleming, P. J., Koppel, D. E., Lan, A. L. Y.,and Strittmatter, P. (1979). lntramembrane position of the fluorescent tryptophanyl residue in membrane-bound cytochrome b,. Biocliemistry 18, 5458-5464. Fox, R. O., Jr., and Richards. F. M. (1982). A voltage-gated ion channel model inferred from the crystal structure of alamethicin at I 5-A Resolution. Nature (London) 300, 325-330.

Franks. N . P. (1976). Structural analysis of hydrated egg lecithin and cholesterol bilayers. I. X-Ray diffraction. J . Mol. B i d . 100, 345-358. Fuldner. H. H. (1981). Characterization of a third lipid transition in multilamellar dipalmitoyllecithin liposomes. Biochemistry 20, 5707-57 10. Galla, H. J . , and Sackmann, E. (1975). Chemically induced lipid phase separation in model membranes containing charged lipids: A spin label study. Bioclzim. Biophvs. Acts 401, 509-529. Golan. D. E., Alecio, M. R., Veatch, W . R. and Rando, R. R. (1984). Lateral mobility of

phospholipid and cholesterol in the human erythrocyte membrane: Effects of proteinlipid interactions. Biochemistry 23, 332-339. Grant, C. W. M.. W u , S. H.-W.. and McConnell, H. M. (1974). Lateral phase separations in binary lipid mixtures: Correlation between spin label and freeze-fracture electron microscope studies. Biochim. Biophys. Acia 363, 151-158. Griffin. R. G . . Powers, L., and Pershan. P. S . (1978). Head-group conformation in phospholipids: A phosphorus-3 I nuclear magnetic resonance study of oriented monodomain dipalmitoylphosphatidylcholinebilayers. Biochemistry 17, 2718-2722.

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Griffith. 0 . H.. Ikhlinger. P. J.. and Van. S. 1’. 11974). Shape o f the hydrophobic barrier o f phospholipid bilayers (evidence for water penetration in hiological membranes). J . M’.s. 185, 21-29.

Gerber. G. E.. Anderegg, R. J., Helihy, W. C.. Gray, C. P.. Biemann, K., and Korana, H. G. f 1979). PWC. NU//. Al.rit/. S(.i. U . S . A . 76, 227-23 I . Gerritsen, W. J.. Verkleij. A. J., and van Deenen. L . 1,. M. (1979). Bicwhirn. Biopl7y.s. Actcr 555, 26-4 I . Gershon, N. D.. Demsey, A.. and Stackpole. C. W. (1979). Exp. Cell Res. 122, 11-126. Giese. G., Fromme. I.. and Wunderlich. F. (1979). Eur. J. Biochrm. 95, 275-28s. Gilula. N. B., Eger, R. R.. and Fifkin. D. B. (1975). Proc. N o t / . Acnd. Sci. U . S . A . 72, 3594-3598.

Goormaghtigh, E.. van den Branden, M., Ruysbchaert. J . M., de Kruijff. B. (1982).Bioclrirn. B i o p h y s . A c ~ 685, ~ I 137-143. Gounaris, K., Brain, A. P. R., Quinn. P. J., and Williams, W. P. (1983a). FEBS Ltrr. 153, 47-52.

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CUKKF.NT‘ropicsI N M E M B K A N E S A N D .IKANSPOKT.

V O L U M E ?Y

The Thermodynamics of Cell Adhesion MICAH DEMBO A N D GEORGE I. BELL lhrorcticcil Division Lo.\ A1trmo.s Nirrionul Lriborirrorv Los Altrtnos. N e w Me.ti1.o 87545

1.

INTRODUCTION

Equilibrium thermodynamics as a rigorous discipline is restricted to the description of completely inanimate or ”conservative” systems. Nevertheless, even in biological systems that are more or less far from equilibrium, thermodynamic models can give us some idea of the natural end point towards which the system would tend if active metabolism were neglected. In the case of cell-cell and cell-surface adhesion the study of thermodynamic models is also important because these models can provide a language for the clarification and integration of a variety of subtle and vaguely defined notions.

II. THEMODEL

In order to illustrate the principles involved in taking a thermodynamic approach to modeling cell adhesion, we will present the detailed formulation and analysis of an elementary example: adhesion of a cell to a large flat surface, herein called the “substrate.” To start with, consider that the cell has “receptors” that can bind, in a lock and key fashion. to complementary ‘‘sites” on the substrate. We will regard the sites as fixed and immobile in the plane of the substrate. In contrast, we will allow the receptors to be freely mobile in the entire plane of the plasma membrane.’

‘ A detailed discussion of a different special case. adhesion of a cell to another cell where receptors on both cells are free to diffuse laterally, appears elsewhere (Bell (’I ( / I . , 1984). 71

Copyright nl 19x7 by Aciidemic Pres*. Inc. All rights of‘ reproduction in a n y form re\erved

72

MICAH DEMBO AND GEORGE I. BELL

In sum, we will view cell adhesion as resulting from the formation of specific receptor-site bonds; the net effect of all other forces between cell and substrate is taken to be repulsive. Given this rather elementary formulation, the next problem is to define precisely what is meant by “contact” between the cell and the substrate (see Fig. 1). To accomplish this, let the surfaces of the cell and the substrate be represented by two sets of points, {A,} and {As},respectively. We now propose that interactions between the cell and the substrate can only occur within a definite subset of “contact points,” {A}. Naturally, inside the set {A} the cell surface and the substrate must be in relatively close proximity, with the average separation distance S. We will regard points on the remainder of the cell surface as being separated from the substrate by distances very much greater than S. Let us denote the total surface density of receptors and sites at a position x by n,(x) and n,,(x), respectively. Since the substrate is a uniform material, and since sites are immobile, it is clear that n,,(x) = n,, must be constant for all x E {As}.Furthermore, because receptors and sites can only be in one of two states, free or attached, we surmise that both total surface densities can be broken into two terms

n,t = n,(x) -t nh(x)

X

E {A,}

(2)

where n,(x) and n,(x) are the surface densities of unattached receptors and sites and &(x) is the local surface density of cell-to-substrate bridges. If we consider the distribution of bridges over the plasma membrane, it is obvious that nh(x) must be zero outside the contact region. On the other hand, since entropy must be maximized near thermal equilibrium, the equivalence of all contact points implies that the density of cell-substrate bridges will be a positive constant for points inside the contact set. Therefore, if A is the area of the set {A}, and N h represents the absolute number of bridges between the cell and the substrate, then near equilibrium the density of bridges on the plasma membrane is a discontinuous function nh(X) = nh

Nb/A

x E {A}

and nh(X) = 0

x E {A,} - {A>

(3a)

Furthermore, inserting this expression into Eq. (2), it is easily seen that the density of free sites on the substrate is also discontinuous

73

THE THERMODYNAMICS OF CELL ADHESION

~I,(x) = n,,

-

x E {A}

n,,

and n,(x)

x E {A,}

= n,,

-

{A)

(3b)

In contrast to sites on the substrate and cell-substrate bridges, unattached receptors are freely mobile. Thus, since free receptors can exist with equal probability at all points on the cell surface, both inside and outside the contact region, we see that close to thermal equilibrium n , ( x ) must have the same density both inside and outside the contact area. Thus, if A, is the area of the set {A,} (i.e., A, is the total cell surface area). then IZ,(X)

= n, =

(A',, - N,)/A,

x E {Ac}

(3C)

We are now in a position to consider the Gibbs free energy of the closed system formed by a cell in contact with a substrate. To this end, let the repulsive force between membrane and substrate, at a separation distance S , be F(S) per unit area of contact. Furthermore, let p,[n,(x)], p,[n,(x)l, and pb[nh(x),S]represent the chemical potentials of receptors, sites, and bridges at an arbitrary position x. Given this notation, the free energy will

Unattached Receptors

Unattached Sites

f

Cell - Substrate Bridges

FIG. I . The model of cell adhesion. A cell is assumed lo possess a fixed number of freely diffusing receptors. whereas the substrate is covered by a constant density of imrnohile sites. Binding between receptors and sites can occur only in a "contact region'' where the separation distance between the two surfaces is small.

74

MICAH DEMBO AND GEORGE I. BELL

be of the form

(4a)

+ constant terms In order of appearance the various terms in Eq. (4a) represent (1) the free energy of unattached receptors on the cell surface; (2) the free energy of unoccupied sites on the substrate; (3) the free energy of cell-substrate bridges; (4) the work that must be done against nonspecific forces in order to bring the cell and substrate to a separation distance of S over an area A; and (5) constant terms representing the free energy of those parts of the system that are unperturbed by the occurrence of contact. Using Eqs. (3a), (3b), and (3c) we can quite simply compute the various integrals in Eq. (4a). When this is done, we obtain the simpler expression G(Nd,S)

=

(Nrt

-Nb)F,[(N,t - Nt,)/A,I

i(Anst -

NtJPJn,, -(NdA)I

+ (A, - A)nst~Jn,t1

(4b)

+ Nbpb(Nb/A,S)+ A r ( S ) + constant terms where A, is the total area of the substrate and z

us) = .I- m d Z S

is the repulsive potential per unit area of contact. When written in the form of Eq. (4b), it is apparent that the free energy can be regarded as a function of only three independent variables: &, A, and S. Thus, by finding the minima of Eq. (4b) with respect to these three variables, we can determine the number of distinct stable states of the cell-substrate system as well as the most probable equilibrium properties of these states. However, in order to proceed with an analysis of the free energy function, we must first obtain explicit expressions for the chemical potentials and for the potential energy of nonspecific repulsion.

111.

THE CHEMICAL POTENTIALS

In the “ideal solution” limit, the chemical potentials of the free receptors and free sites can be related to the corresponding concentrations according to the usual expressions

75

THE THERMODYNAMICS OF CELL ADHESION

and

pr(n,)= p:!

+ kT t‘n(n,)

(Sa)

k,(n,) = ky

+ k7’ tn(n,)

(Sb)

Notice that although the receptors are freely mobile and the sites are fixed in space, this makes no difference as far as the form of the expressions for the chemical potential is concerned. The chemical potential of a cellsubstrate bridge is only slightly more complicated: pLh(nh,S) = pE(S) + k’l’ h(n,,)

(5c)

In Eqs. (5aL (5bL and (5c), p:, p:‘, kE(S) represent the chemical potentials of receptors, sites, and bridges at a “standard” concentration (e.g., one molecule per pm’). k: and p! are simply constants because the internal energy of unattached states is not affected by the amount of cell-substrate separation. In contrast, pE(S) depends quite critically on the cell-substrate separation because changes in this distance result in stretching or compression of the bridges (see Fig. 2). If we think of a bridge as having a certain amount of elasticity, then we can characterize the S dependence of pE(S) in terms of the “resting” or unstressed length of the bridge, L ,

0

s =L

1

FIG.2. The chemical potential of cell-substrate bridges is dependent on the separation distance. I f S is too small. as in A, then the chemical potential is large because the bridges are compressed. If S is too large. as in C . then the chemical potential is large because the bridges are stretched. There is thus a certain value of the separation distance. .Y = 1.. such that the chemical potential of cell-substmte bridges is minimal ( B ) .

76

MICAH DEMBO AND GEORGE I. BELL

and a force constant for stretching of the bridge, K. In terms of these parameters, the chemical potential of a unit concentration of bridges is

Cl”,(s)=

&(L)

+ i K ( S - L)’

-k

...

(6)

In subsequent developments, it will be useful to define the idea of an equilibrium constant for cell-substrate bridging at a given separation distance. By direct analogy with the classical equilibrium constant of solution phase reactions, we will therefore introduce K ( S ) = exp [

( ~ y+

p! - pt(S)

+ kT)/kT]

(7a)

Inserting Eq. (6) into (7a) we see that to a good approximation K ( S ) = K , exp [ - ~ K ( S- L)’/kT]

(7b)

where KL = K f L ) represents the equilibrium constant for formation of an unstressed bridge. IV. THE REPULSIVE POTENTIAL

The repulsive potential, T(S),is defined as the mechanical work that must be done against nonspecific forces in order to bring a unit area of membrane from an infinite separation to a separation of S. If the repulsive force between unit areas of membrane and substrate is F(S), then F(S)

=

-rl(s)

or equivalently

r(s)= 7 m ) d z S

According to recent estimates (Bongrand and Bell, 1984), the forces resisting adhesion arise mainly from a combination of two effects: (1) electrostatic repulsion between negative chafges associated with the surfaces, and (2) the so-called steric stabilization effect.* The steric stabilization force is well known in the theory of colloid suspensions (Napper, 1977); it arises because cell membranes are coated by hydrated layers of long chain polymer molecules (i.e., the glycocalyx). In many systems the substrate could also be coated by a layer of absorbed polymers (e.g., serum proteins); this might enhance repulsive effects somewhat, but it is not an essential factor for the steric stabilization force. As the cell approaches *Since cell surfaces are generally negatively charged, it is possible to obtain an attractive electrostatic force to a positively charged substrate, e.g., one coated with polylysine. However, in this article we assume the net force to be repulsive.

77

THE THERMODYNAMICS OF CELL ADHESION

A

0

s< T FIG.3. The repulsive force between cell and substrate. If the separation distance is much larger than the average thickness of the glycocalyx ( A ) . then the repulsive force will be negligible. If the separation distance is less than the average thickness of the glycocalyx (B), then several factors will lead t o repulsive forces. There are ( 1 ) the osmotic tendency of solvent to return into a region of high polymer concentration. ( 2 ) the steric compression of the polymer chains, and ( 3 ) electrostatic repulsion between fixed charges on the substrate (if they are negative) and the glycocalyx.

the substrate, the polymer molecules of the glycocalyx are compressed between rigid surfaces, and in addition, water of hydration is squeezed out of the cell-substrate gap (see Fig. 3). The repulsive force results partly from the steric compression of the polymers and partly from the osmotic pressure caused by the tendency of solvent to return into the gap. An exact description of the complex interactions that go into determining the detailed form of electrostatic and steric stabilization forces are beyond the scope of this article. Instead, we will adopt a simple phenomenological expression for US) that has the correct qualitative behavior,

U S ) = -YS exp ( - S I T )

(8)

From a descriptive point of view, the parameter y measures the stiffness of the glycocalyx; the larger the value of y the harder it is to compress the polymers. The parameter T measures the thickness of the glycocalyx. If S is much greater than T , then there is a negligible compression of the glycocalyx; if S is much less than T , then the glycocalyx is compressed to the point where it fills the entire cell-substrate gap uniformly (i.e., the thickness of the gap is much smaller than the thickness of the glycocalyx). Typical values of T for cell-substrate interactions are in the range of SI S nrn.

78

MICAH DEMBO AND GEORGE I. BELL

Theoretical estimates of y can be derived by considering the statistical mechanics of chain molecules anchored to rigid surfaces. For typical cell parameters such estimates yield values of y on the order of lo-‘ to lo-’ dynes (Bongrand and Bell, 1984). The contribution of electrostatic repulsions to the value of y can also be estimated on the basis of theoretical argument. However, it seems that electrostatic effects do not significantly add to the value of y unless the glycocalyx is very sparse or the charge density is unusually high (Bongrand and Bell, 1984).

V.

MINIMIZING THE FREE ENERGY

Returning to the process of finding the minimum value of the free energy with respect to N b , A, and S, we should first note that none of these variables has a completely unrestricted range. For example, since the separation distance is obviously strictly positive, we must require that (94

OSSSCO

In a similar way, it is obvious that the number of cell-substrate bridges cannot increase if there are no free receptors left on the cell or if all the sites on the substrate are already occupied. Thus the number of bridges must fall in the range 0 s N , s min [N,,An,J

(9b)

Finally, the area of cell-substrate contact cannot increase if the cell has become completely flattened out to the point where there are no further reserves of “slack” membrane. As indicated by Fig. 4, if the surface-tovolume ratio of the cell is small, then the maximum possible amount of spreading will be small; if the surface-to-volume ratio is large, then the maximum contact area will approach ‘LAc. In any event, the contact area will, in general, be subject to a constraint of the form

0 > U . They showed that the time for encounter between diffusing molecule and target was proportional to a tracking factor, .f(blcr). The factor was linear in hltr for three-dimensional diffusion, was proportional to log hlu in two dimensions, and was independent of hln in one dimension. They argued that two-dimensional diffusion or combinations of three- and two-dimensional diffusion would result in significantly shorter "catch" times than would threedimensional diffusion alone. The biological example used was the catch of pheromones on the waxy cuticle surrounding specific pheromone sensors of moths. This evaluation was carried further by Hardt (19791, who showed how the concentration of interacting species affects reaction rates, and showed that the sensitivity of reaction rates to concentrations of reactants increases with decreasing dimensionality. The first general suggestions that diffusion might couple reactants came from work on activation of adenylate cyclase by agonist-receptor complexes. The stoichiometry of activation indicated that a single pool of enzyme was activated by many different receptor-agonist complexes, and this was best accommodated by a model in which receptors encountered cyclase at random by diffusion (Cuatrecasas, 1974; De Haen, 1976). It was later shown that receptors implanted in membranes by cell fusion or by partion from detergent extracts could functionally activate adenylate cyclase of the recipient cells (Orly and Schramm, 1976; Eimerl of d., 1980). further implying that receptor and enzyme interacted by random collision. Hanski et a/.(1979) were able to show that reducing membrane viscosity by incorporation of cis unsaturated fatty acids increased the rate of interaction about 2-fold, and they derived a plausible diffusion coeficient for the receptor and/or the cyclase: D -4 x lo-" cm' sec-'. However, the model is complicated by the demonstration of a coupling protein, the GTP-regulatory protein, that mediates the interaction of receptors with

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MICHAEL EDlDlN

adenylate cyclase (for review see Levitski, 1981). The coupling protein need not be integral to the membrane bilayer and may diffuse on the inner surface of the bilayer. Thus, while the evidence for lateral diffusion coupling of all reactants remains strong, the diffusion coefficient derived for the reaction needs to be reevaluated. One other interaction between components of the plasma membrane appears likely to be mediated by lateral diffusion: the entry of low density lipoprotein (LDL) receptors into coated pits. Goldstein et af. (1981) used published data on the lateral diffusion rate of LDL receptors, -lo-" cm' sec-', and on the surface density and metabolism of coated pits and receptors to derive a plausible model for the entry of receptors into coated pits by lateral diffusion. This analysis might be generalized to all receptors which internalize through coated pits. Two studies suggest that components of electron transfer systems in endoplasmic reticulum interact by diffusion and random collision (Yang, 1977; Strittmatter and Rogers, 1975). However, these studies are based on interactions in reconstituted model membranes. The most thorough and convincing series of papers on diffusion coupling of reactions comes from Hackenbrock and co-workers, who have been able to relate lateral diffusion and coupling in electron transport chains of mitochondria1 inner membranes. The work involved the demonstration of lateral diffusion of membrane particles quoted earlier (Sowers and Hackenbrock, 198 I), the creation of lipid-enriched mitochondria which allowed study of the dependence of redox reactions on the density of donors and acceptors, and the use of FPR and a variety of specific fluorescent labels to study lateral diffusion of cytochrome b-c,, cytochrome oxidase, cytochrome c, and ubiquinones in inner membranes of giant mitochondria (Gupte et a/., 1984). The integral membrane proteins cytochrome b-c, and cytochrome oxidase diffused at around 4 x lo-"' cm' sec-', somewhat more slowly than estimated for typical membrane proteins by Sowers and Hackenbrock (1981), while a ubiquinone analog and a lipid-soluble dye diffused 6 and 10 times faster, respectively. Cytochrome c , a peripheral protein, diffused at a rate dependent on ionic strength, being almost immobile in low ionic strength medium and diffusing at around 2 x cm' sec-' in 25 mM saline buffer. Recovery of cytochrome L' fluorescence after bleaching appeared to be due to a mixture of two- and three-dimensional diffusion. Recoveries of the other labeled components were around 90%. Thus recoveries are as high as those found for proteins in synthetic or cytoskeleton-free membranes, while the diffusion coefficients, even for the lipid probes, are about an order of magnitude slower than seen in synthetic membranes. Much of this difference must be due to the high protein concentration in the inner membrane. Effects of membrane protein concentration on lateral diffusion have been discussed above.

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119

The diffusion coefficients estimated for complexes I-IV from these measurements were used to calculate collision frequencies for redox components, and these were found to be greater than the maximum turnover numbers observed for all redox components. Thus lateral diffusion rates are sufficient to couple all components in mitochondrial electron transport, which then must be randomly arranged in the mitochondrial membrane. Here it seems we have found a membrane in which lateral diffusion serves effectively to organize function.

V.

A CONCLUDING REMARK

I have tried to use a limited number of examples to cover the methodology and results for rotational and lateral diffusion of membrane proteins. I hope that this article has shown some directions in which the measurements of diffusion may be taken to shed light on membrane function.

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Thomas. D. D.. Carlsen. W . F . , and Stryer. L. (1978). Fluorescence energy transfer in the rapid diffusion limit. Proc. N o r / . A(,crd. .Sc.i. O . S . A . 75, 5746-5750. Vanderkooi. J . M.. and Callis, J. B . (19741. Pyrene. A probe o f lateral diffusion in the hydrophobic region of membranes. Biochc~vii.yti:\.13, 4(K)O. van Meer. G . , and Simons, K. (1982). Viruses budding from either the apical or the basolateral plasma membrane domain of MDCK cells have unique phospholipid composttionc. E M U 0 J . I , 847-852. van Zoelen. E . J . J.. Tertoolen. L. G . J.. and de Laat. S . W. (1983). Simple computer method for evaluation of lateral diffusion coefficients from fluorescence photobleaching recovery kinetics. Biopliys. J . 42, 103-108. Vaz. W., Austin. R. H.. and Vogel. H . (1979). The rotational diffusion of cytochrorne h, in lipid bilayer membranes. B i o p h ? ~J. . 26, 415426. V u . W. L. C.. Criado, M., Madeira. V. M. C.. Schoellmann. G . . and Jovin. T. M. (1982). Size dependence of the translational diffusion of large integral membrane proteins in liquid-crystalline phase lipid bilayers. A study using fluorescence recovery after photohleaching. Bioc./imiisrn 21, 5608-561 2. Wagner. R.. Carrillo. N., Junge, W . . and Vallejos. R. H . (1982). On the conformation of reconstituted ferredoxin:NADP' oxidoreductase in the thylakoid membrane. Studies via triplet lifetime and rotational diffusion with eosin isothiocyanate label. L i i o c , / i i r t t . Riop/rys. Ac/tr 680, 3 17-330. Wey. C.-l.. Ahl. P.. Cone, R. A , , and Ciaffney. B . J . (l979j. Membrane viscosity-ii small lipid probe reports the same viscosity iis two integral membrane proteins. Riophys. J . 25, 169a. Wey. C-I,, R. A,. Cone. and Edidin. M. A . (1981). Lateral diffusion of rhodopsin in photoreceptor cells measured by fluorescence photobleaching and recovery. Biopfty.y. J . 33, 225-232. White, R. E.. and Coon. M. J . (1980). Oxygen activation by cytochrome P-450. Annrr. HPI.. Bioc~//cm.49, 3 15-3.56. Wolf. D. E . . Edidin. M. A,. and Drngsten. P. K. (1980). Effect of bleaching light on nieasurements of lateral diffusion in cell membranes by the fluorescence photobleaching recovery method. Proc. Ntirl. Acntl. S c i . U . S . A . 77, 2043-2045. Wu. E.-S.. Tank, D. W . . and Webb, W. W. (1982). Unconstrained lateral diffusion of Con A receptors on bulbous lymphocytes. Pro(.. Nctrl. A(,cid. .%i. U . S . A . 79, 4962-4966. Yang. C. S. ( 1977). Organization and interaction o f monooxygenase enzymes in the microsoma1 membrane. k f i , Sci. 21, 1047-10.57. Zagyansky. Y . , and Edidin, M . (19761. Lateral diffusion ofconcanavalin A receptors in the plasma membrane in mouse fibroblasts. B i o ( ~ / i i v iBiop/iys. . Actci 433, 209-214. Zagyansky, Y ., and Jard. S. (1979). Does lectin-receptor complex formation produce Lone\ of restricted mobility within the membrane? Ntrtirrc f L o n t h r i ) 280, 591-593. ZidovetLki. R . , Yarden, Y.. Schlessinger, J . . and Jovin, T. M. (19x1). Rotational diffusion of epidermal growth factor complexed to cell surface receptors reflect\ rapid niicroaggregation and endocytosis of' occupied receptors. Proc.. Nor/. Accrd. Sci. U . S . A . 78, 698 1-6985.

Ziomek. C. A , . Schulman. S., and Edidin. M. (1980). Redistribution of membrane proteins in imlated mouse intestinal epithelial cells. J . Cell B i d . 86, 849-857,

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C U R R h N T IOPICS IN MkMBRANLS A N D I R A N S P O R T . VOLUME 29

Biosynthesis and Distribution of Lipids K E N N E T H J . LONGMUIR Di,pcirtrnent of Physiology (rnd BiopliysicJ Ctrlifornia College of Medicine Llnit~ersitvof California. Irvine Irvine. Cdifornirr 92717

1.

INTRODUCTION

Membrane biogenesis in eukaryotic cells is an important topic of research in contemporary cell biology. This problem is being successfully investigated largely by studies which explore the intracellular transport of newly synthesized, membrane-bound protein (Sabatini et n l . , 1982). Current models suggest that polypeptides, synthesized on ribosomes in the endoplasmic reticulum, move through the endoplasmic reticulum and Golgi apparatus, where they are sorted and targeted to other regions of the cell such as the plasma membrane, lysosomes, or secretory organelles. A less conclusive picture is available for the synthesis, transport, and targeting of membrane lipids in eukaryotic cells. This lack of information is due partially to the experimental difficulties encountered when attempting to follow subcellular synthesis and transport of lipid. Whereas antibodies and lectins are successfully used to determine the location of intracellular proteins, these methods are not generally available to the lipid biochemist. Interpretation of data obtained by subcellular fractionation methods is often limited by contamination of one subcellular membrane fraction by another. Autoradiography of lipids in cells is difficult unless care is given to the retention of lipid during tixation, dehydration, and embedding procedures. Despite the experimental limitations the subcellular sites of lipid synthesis, the intracellular lipid transport processes, and the targeting of lipids to specific membranes are recognizcd as important topics of current interest to lipid researchers. As a result, considerable data have accumulated in recent years which address these subjects. This article provides an overview of lipid metabolism in mammalian 129 Copyrighi 'C' 1987 hy Academic h e $ \ . Inc.

All righlr 01' repr(iduction

in

any forin rcaeivcd.

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KENNETH J. LONGMUIR

cells from the point of view of subcellular synthesis, transport, and targeting of lipid. It discusses the available data which indicate that different subcellular membranes have, to some extent, different lipid compositions. It discusses the various biosynthetic pathways of fatty acid and phospholipid biosynthesis, with emphasis placed on the subcellular locations of the biosynthetic enzymes. Finally, this review discusses the possible mechanisms responsible for the transport of lipid between subcellular membranes and the mechanisms by which cells produce and maintain membranes of different lipid compositions. This article covers a wide range of topics and is intended as an overview for those not familiar with lipid biosynthesis. Some areas are not considered because they are not pertinent to the subject of membrane biogenesis or because excellent reviews have appeared in recent years. For example, fatty acid biosynthesis has been reviewed recently (Wakil et al., 1983), and only fatty acid desaturation and elongation reactions are considered here. Also, space does not permit discussion of glycolipid biosynthesis, nor of the interesting changes in lipid biosynthesis that occur under hormonal or developmental control. Excellent reviews have appeared on nearly all of the topics in this article, and these are cited in the individual sections. II. THE LIPID COMPOSITION OF SUBCELLULAR MEMBRANES A. Nomenclature 1. FATTY ACIDS

Table I contains the names of the principle fatty acids esterified to the lipids of mammalian tissue. The list is limited to those fatty acids which are present in amounts greater than a few percent in most tissues or which are major products of fatty acid biosynthetic pathways. Because fatty acids have long systematic names, a shorthand notation is used to indicate the number of carbon atoms followed by the number of cis (or Z) double bonds. Hence palmitic acid is 16:O and arachidonic acid 20:4. There are two conventions for designating the locations of double bonds. One numbers from the carboxyl end, the other (the o nomenclature) from the terminal methyl group. Linoleic acid, for example, is designated 18:2 (9,12) when numbering from the carboxyl end, and 18:2 w6,9 when numbering from the terminal methyl group. The w nomenclature is useful because of the nature of fatty acid desaturation and elongation reactions in mammalian tissue. Desaturation occurs between an existing double bond and the carboxyl end. Elongation reactions occur at the carboxyl end. As a result, despite the many com-

131

BIOSYNTHESIS AND DISTRIBUTION OF LIPIDS

TABLE 1 PRINCIPAL FATTYACIDS01 M A M M A L I ATISSUE. N Common name

Systematic name

Myristic Palmitic Stearic Palmitoleic Oleic Linoleic Linolenic Arac hidonic

T,etradecanoic Hexadecnnoic Octadecanoic 9-Hexadecenoic 9-Octadecenoic 9.12-Octadecadienoic

-

4.7.10.13.I6.19-Doco\ahexaenoic

“

9,12.15-Octadecatrienoic 5 . 8 , I I , 14-lcosatetraenoic

Shorthand notation 14:O 16:O IX:0 I 6 : I (9)” I8:I (9) 18:2 (9.12)

18:3 (9,12,15) 20:4 (5.8.1 1.14) 22:6 (4,7,10,13.16,19)

All double bonds are cis (or Z ) and are numbered from carboxyl end.

binations of desaturation and elongation reactions that take place, the number of carbon atoms between the terminal methyl group and the nearest double bond remains constant for a given family of fatty acids. Oleic acid and the products of desaturation and elongation of oleic acid are the (119 ) or w9 fatty acids. Palmitoleic acid and its products are (n-7) or w7 fatty acids. Linoleic acid and arachidonic acid are both w6 or (n-6)fatty acids. Linolenic acid and its products are the w3 or the ( n - 3 ) family of fatty acids. [The principal desaturation and elongation pathways of mammalian tissues are listed in Section I11 (Table 1111.1 Double bonds of polyunsaturated fatty acids are separated by one methylene carbon. Using the w nomenclature, it is necessary to specify only the position of the double bond nearest the terminal methyl group. Using this convention, arachidonic acid can be specified as 20:4 06 (which can be written out as 20:4 w6,9,12, IS).

2. GLYCEKOLIPIDS Glycerolipids share the common feature of the glycerol backbone, which in biological systems becomes stereospecific when esterified with phosphate groups, fatty acids, or both. Their structures are best indicated using the stereospecific numbering system (Hirschmann, 1960; IUPAC-IUB, 1978) which has become widely accepted for the naming of glycerol derivatives of biological interest. With a few exceptions (see Section IV,E,S), glycerolipids are products of sn-glycerol 3-phosphate. The phosphate group is found at s n - 3 , while fatty acids are esterified to sn-1 and sn-2 (Fig. I , A). The particular class of glycerophospholipid is determined by the nature of the polar head group (Fig. 1. B).

132

KENNETH J. LONGMUIR

A sn- 1

sn-2 I

I 0 I II HZC-0-P-X

sn-3

I

0B Phosphatidic a c i d

-OH

Phosphatidylcholine

Phosphatidylethanolamine

coo -

Phosphatidylserine

-0-CH

-CH,

/

~

NH3

OH

OH

Phosphatidylinositol

-OQOHOH

Phosphatidylglycerol

-0-CH,-CH-CHI I

I

OH OH

FIG.I . ( A ) Stereochemistry of sn-glycerol3-phosphate and its glycerophospholipid products. X = polar head group. (B)Names and polar head groups of the principal glycerophospholipids derived from phosphatidic acid.

The parent compound of the glycerolipids is phosphatidic acid. If ambiguities arise, the position of the phosphate group on the glycerol backbone may be indicated (e.g., 3-sn-phosphatidic acid) or it may be specified by using the systematic name, 1,2-diacyl-sn-glycerol 3-phosphate. When the fatty acid composition is known, it may be included (e.g., I-stearoyl2-oleoyl-sn-glycerol 3-phosphate). Glycerophospholipids are named most often as derivatives of phosphatidic acid, hence the terms phosphatidylcholine, phosphatidylethan-

BIOSYNTHESIS AND DISTRIBUTION OF LIPIDS

133

olarnine. phosphatidylserine, phosphatidylinositol, and phosphatidylglycerol. The location of the phosphate group is usually not indicated but for the most part is assumed to be srr-3. When necessary, the position of the phosphate may be indicated (e.g., 3-sii-phosphatidylcholine) or the name may be written out to the known degree of specificity (e.g., 1,2diacyl-sn-glycero-3-phosphocholineor I-palmitoyl-2-linoleoyl-sn-glycero3-phosphocholine). Some glycerolipids in mammalian tissue (the “ether lipids”) contain a hydrocarbon chain attached to the glycerol backbone by an ether linkage at position s n - I , with a fatty acid esterified to position sn-2. If a double bond is not present at position I ’ of the hydrocarbon chain, these are named I-alkylglycerolipids. When the alkyl and acyl chains are known, their names may be included, such as I-hexadecyl-2-oleoyl-stl-glycero-3phosphoethanolamine. A second class of ether lipids are those which have a cis (or Z ) double bond between carbons 1’ and 2’ of the chain at position s t i - I . They are called I-( I ‘-alkenyl)-2-acylglyccrolipids, but are usually referred to by their common name, plcrsmrrlogetzs. Choline plasmalogens are I-( I ’-alkenyl)-2acyl-.s~~-glycero-3-phosphocholines, and ethanolamine plasmalogens are I ( I ’-alkenyl)-2-acyl-sn-glycero-3-phosphoet hanolamines. Finally, the designation 0 indicates that the fatty acid or fatty alcohol is linked via the oxygen atom to the glycerol backbone. The proper designation for phosphatidic acid is therefore I -O-acyl-2-O-acyl-sn-glycerol 3-phosphate. It is permissible to omit the 0 designation. as is usually done.

6. Phospholipid Composition This section provides an overview of the phospholipid and fatty acid compositions of the subcellular membranes of mammalian tissue. Studies of phospholipid composition are dependent, of course, upon successful separation of tissue into the various subcellular fractions in vitro. For mammalian tissue, the best data are for the liver, as the methods for obtaining clean subcellular fractions are more advanced for liver than for other tissues. Prominently cited data of the phospholipid compositions of liver subcellular fractions are the reviews of McMurray and Magee (1972), McMurray (1973), and the data tables of White (1973). Except for the lysosomal membrane, the data compiled by McMurray and Magee (1972) have been adapted here in Table 11. Data for the lysosomal membrane are from the work of Bleistein rr ul. (1980). It must be cautioned that one relies upon the combined data of several investigators to obtain an overview of the compositions of subcellular membranes, even for liver tissue.

TABLE I1 PHOSPHOLIPID COMPOSITION OF SUBCELLULAR MEMBRANES FROM RAT LIVER" Mitochondriab Phospholipid Phosphatidylcholine Sphingomyelin Phosphatidylethanolamine Phosphatidylserine Phosphatid ylinositol Phosphatidylgl ycerol Cardiolipin Phosphatidic acid Lysophosphatidylc holine L ysophosphatid ylethanolamine Bis(monoacylg1ycero)phosphate

Inner membrane

Outer membrane

Nuclear membranes'

Rough endoplasmic reticulumd

Golgi membranesd

Plasma membrane'

Lysosomal membranes'

45.4 2.5 25.3 0.9 5.9 2. I 17.4 0.7

49.7 5.0 23.2 2.2 12.6 2.5 3.4 1.3

61.4 3.2 22.7 3.6 8.6

60.9 3.7 18.6 3.3 8.9

45.3 12.3 17.9 4.2 8.7

34.9 17.7 18.5 9.0 7.3 4.8

41.6 9. I 27.3 -

-

1 1.5

5.9

6.3

9.4

-

4.4 3.3

1.9

1.3 4.0

' Except for lysosomal membrane data, table adapted from McMurray and Magee (1972). Reproduced. with permission, from the Annird Review ofBiochernistry. Vol. 41 0 1972 by Annual Reviews Inc. Values are expressed as the percentage of the total phospholipid in a given fraction. Where no value is indicated, the phospholipid was either not reported or below the limit of detection. McMurray and Dawson (1969). ' Kleinig (1970). Keenan and Morre (1970). ' Ray e t a / . (1969). Bleistein et al. (1980). Unidentified phospholipid amounted to 3.2% of the total.

BIOSYNTHESIS AND DISTRIBUTION OF LIPIDS

135

Variations occur with the purity of subcellular fractions and with the separation techniques used. As a result, strict comparisons among the various columns of data in Table I 1 are difficult. Only general trends should be observed. Those trends that in the opinion of this reviewer seem most clearly accepted are discussed below.

I . NITKOGEN-CONTAINING PHOSP~IOI.IPIDS a. P h o s p h c i t i ~ y l c h o l i n e . Phosphatidylcholine is the most abundant glycerophospholipid of mammalian tissue. It is found in the greatest proportion in nuclear membranes and in endoplasmic reticulum. The current view of membrane biogenesis is that membrane formation begins in the endoplasmic reticulum. Membrane material then proceeds through the Golgi apparatus to the plasma membrane (Sabatini ef a l . , 1982). The percentage of phosphatidylcholine decreases as this transition is followed. in in the order nuclear membrane = endoplasmic reticulum > Golgi apparatus > plasma membrane. An intermediate level of phosphatidylcholine is found in the mitochondria. On a percentage basis, there is less in the mitochondria than in the nuclear membranes and the endoplasmic reticulum but more than in the plasma membrane. h. S p h i n g o m y e l i n . Sphingomyelin (Fig. 2 ) is most abundant in the plasma membrane. It is present up to a few percent in the mitochondria, if at all, as some investigators do not detect sphingomyelin in clean mitochondrial fractions from rat liver (Fujiki el NI., 1982). The sphingomyelin content of subcellular membranes follows the progression nuclear membrane = endoplasmic reticulum < Golgi apparatus < plasma membrane. The relative proportions of phosphatidylcholine and sphingomyelin vary from tissue to tissue. However, it is noted that for most tissues the amount of choline-containing phospholipid (phosphatidylcholine + sphingomyelin) is relatively constant at approximately one-half of the total phospholipid (reviewed by Barenholz and Thompson, 1980). c . Phosphutidylethunolarninr. Phosphatidylethanolamine is the second most abundant phospholipid of cell membranes. It is enriched in the mi-

l

c=o I

R

FIG.2 .

Sphingomyelin.

136

KENNETH J. LONGMUIR

tochondria. Otherwise, it appears to be nearly equally distributed among subcellular membranes. d. Phosphafidylserine. Phosphatidylserine is a minor lipid component of cells, with the exception of brain tissue, where it can amount to 15% of the total lipid (Baranska, 1982). In liver, the proportion of phosphatidylserine is lowest in mitochondria, intermediate in endoplasmic reticulum and Golgi, and highest in the plasma membrane. 2. ACIDICPHOSPHOLIPIDS a. Phosphatidylinosifol. Phosphatidylinositol makes up approximately 5-10% of the phospholipid of liver membranes, with the exception of the inner mitochondria1 membrane, where the phosphatidylinositol content is low. Otherwise, there is little indication of a preferential enrichment of phosphatidylinositol in any one subcellular fraction. b. Phosphatidylslycerol. Phosphatidylglycerol is present up to a few percent in mitochondria, where it is a substrate for cardiolipin biosynthesis. By some reports, it is present in the plasma membrane. Phosphatidylglycerol is present in a strikingly large proportion (up to 10% of the total lipid) in pulmonary surfactant, where it is the second most abundant phospholipid (Sanders, 1982). c. Cardiolipin. Cardiolipin (Fig. 3 ) is found in the mitochondria only. It occurs principally in the inner membrane, where it is approximately 15-20% of the total inner membrane phospholipid (reviewed by Ioannou and Golding, 1979). The amount of cardiolipin normally present in the outer mitochondrial membrane is not clear, as available data are quite variable. d . Bis(monoacy1glycerolhosphate. Bis (monoacylglycero) phosphate (Fig. 4, also called 1ysobisphosphatidic acid) is localized exclusively to the lysosome. Detection of bis(monoacylg1ycero)phosphate in mitochondria appears to result from lysosomal contamination (Bleistein et al., 1980).

::

0 I1

R,-C-0-CH,

H2C-O-C-R4

HC-O-C-R3

I

0-

I

OH

b-

FIG. 3. Cardiolipin (diphosphatidylglycerol).

137

BIOSYNTHESIS AND DISTRIBUTION OF LIPIDS

0

0 II

II

R,-C-0-CH,

H,C-O-C-R,

I

I

HO-CH

HC-OH

0Flcj. 4. Bisf monoacylg1ycero)phosphate (lysobi~phosphatidicacid).

3. CHOLESTEROL

Available data clearly indicate differences in cholesterol content among the various subcellular membranes although numerical data are somewhat variable. The numbers that follow are from the work of Colbeau et af. (1971). The inner mitochondrial membrane has the lowest cholesterol content (less than 0.05 kmol cholesterol per pmol lipid phosphorus). The outer mitochondrial membrane and the microsomes have similar cholesterol contents (-0.1 pmol/ynol). However, when microsomes are fractionated to yield smooth and rough endoplasmic reticulum, it is found that smooth endoplasmic reticulum contains more cholesterol (-0.24.3 p.mol/ pmol) than rough endoplasmic reticulum (-0.06 p m o l / ~ m o l ) Plasma . membrane contains by far the most cholesterol (0.76 pmol/p.mol). 4. GENERAL OBSERVATIONS A few remarks should be made about the various subcellular membranes themselves. The inner and outer nuclear membranes have similar lipid compositions (Virtanen et al., 1977). Smooth and rough endoplasmic reticulum have similar lipid compositions (Glaumann and Dallner, 1968). I n contrast, inner and outer mitochondrial membranes differ in several respects. The outer membrane is characterized by amounts of phosphatidylinositol and phosphatidylserine similar to the endoplasmic reticulum (Cobleau et al., 1971). The inner membrane has less phosphatidylinositol and phosphatidylserine than other membranes, a higher content of phosphatidylethanolamine, and it contains most of the cellular cardiolipin. Most of the trends concerning the subcellular distribution of phospholipids in liver hold true for the subcellular membranes of other tissues (White, 1973). However, a few prominent differences should be noted. Brain tissue contains more phosphatidylserine and sphingomyelin than most other tissues due to enrichment in myelin. Brain tissue also contains

138

KENNETH J. LONGMUIR

a substantial portion of ethanolamine phospholipid as ethanolamine plasmalogen (McMurray, 1973). Kidney tissue is rich in ethanolamine plasmalogen (Yeung and Kuksis, 1974). In heart tissue, a substantial portion of both the choline and ethanolamine phospholipids are present as choline and ethanolamine plasmalogen. Heart tissue contains about 2-fold more cardiolipin than other tissues, owing to the abundance of mitochondria in cardiac muscle (McMurray, 1973). The phospholipid compositions of subcellular membranes of some mammalian cells in culture have been studied also. The best data are for the BHK-21 cell line, and the subcellular distribution of lipids in this cell line closely corresponds to the distribution found in mammalian tissue (Brotherus and Renkonen, 1977). In contrast, there are reports that some cell lines exhibit abnormal subcellular phospholipid compositions. Bergelson et al. (1974) have reported that some hepatoma cell lines contain (1) significant cardiolipin in subcellular membranes other than the mitochondria, (2) an abnormally large percentage of sphingomyelin in the intracellular membranes, and (3) a higher ratio of phosphatidylethanolamine to phosphatidylcholine in the microsomal fraction. Other investigators have not substantiated the presence of cardiolipin in extramitochondrial membranes of the hepatorna cell lines. However, they have confirmed the abnormally high intracellular sphingomyelin content and the slightly altered phosphatidylethanolamine to phosphatidylcholine ratio in the rnicrosomal membranes (Hostetler et al., 1976, 1979). C. Fatty Acid Composition

Most phospholipid molecules contain a saturated fatty acid at position sn-1 and an unsaturated fatty acid at position sn-2. Most triacylglycerol molecules also contain a saturated fatty acid at position sn-1 and an unsaturated fatty acid at sn-2. In liver tissue, position sn-3 of triacylglycerol contains principally unsaturated fatty acid (Akesson, 1969).

The profile of fatty acids found at each position depends on the class of lipid. In rat liver, triacylglycerol contains substantial amounts of palmitate (16:O) at sn-l and oleate [18: l (9)] or linoleate [ 18:2 (9,12)] at sn2. Triacylglycerol contains less stearate ( N O ) and arachidonate [20:4 (5,8,11,14)] than is found in the glycerophospholipids (Akesson, 1969). Phosphatidylethanolamineand phosphatidylcholine contain significant amounts of all the major fatty acids. Palmitate (16:O) and stearate (18:O) are abundant at s n - I , whereas oleate [18:1 (9)], linoleate [18:2 (9,12)], ara c h i d o n a t e [20:4 ( 5 , 8 , 1 1 , 1 4 ) ] , and d o c o s a h e x a e n o a t e [22:6 (4,7,10,13,16,19)] are found at sn-2. Phosphatidylinositol and phosphatidylserine have relatively low contents

BIOSYNTHESIS AND DISTRIBUTION OF LIPIDS

139

of palmitate, oleate, and linoleate. Instead, stearate is usually found at sn-1 and arachidonate at sn-2 (Holub and Kuksis, 1978). Cardiolipin is equally unusual. In mammalian tissue. it contains upward of8094 linoleate (loannou and Golding, 1979). Nonrandom pairings of fatty acids have been reported (reviewed by Holub and Kuksis, 1978). In liver, preferen.tial pairings of palmitate with linoleate and stearate with arachidonate are noted, although these pairings are far from exclusive. Pairing of fatty acids is most evident in phosphatidylinositol, where in liver approximately 80% of the total phosphatidylinositol consists of stearate at position s n - I and arachidonate at position SH-2.

Differences in fatty acid composition among subcellular membranes are less evident than differences among phospholipid classes. For liver tissue, no clear differences i n the fatty acid profiles of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine are found between the mitochondria lipids and those of the endoplasmic reticulum (Colbeau et a / . . 1971; Holub and Kuksis, 1978). However, it is generally agreed that the plasma membrane contains a greater percentage of saturated fatty acid than the intracellular membranes such as the endoplasmic reticulum and the mitochondria (Keenan and Morre, 1970: Cobleau r f d., 1971). Golgi apparatus appears to contain a level of saturated fatty acid between that of the endoplasmic reticulum and the plasma membrane. There are two aspects to this enrichment with saturated fatty acid. First, the major phospholipids (phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine) contain a higher percentage of saturated fatty acid in the plasma membrane than in the intracellular membranes (Keenan and Morre, 1970). Second, plasma membrane is enriched with sphingomyelin. Sphingomyelin has a higher percentage of saturated fatty acid than most of the other phospholipids (White. 1973). Some tissue-specific differences should be noted. Lung surfactant, a lipoprotein material secreted by a lung alveolar cell, is highly enriched with palmitate at both the sn-1 and sn-2 positions of phosphatidylcholine (Sanders, 1982). It is this saturated phosphatidylcholine which gives surfactant its remarkable surface tension lowering properties. The lipid of brain tissue is also enriched with saturated phosphatidylcholine (though not to the extent found in lung) because of its abundance in myelin. In contrast, lipids of reproductive tissue are notably enriched with ii variety of polyunsaturated fatty acids (Kuksis, 1978). Products of the linoleate series (the 0 6 family of fatty acids) include not only arachidonate [20:4 (S,8,11,14)]. but also 20:3 (8,11,14), 22:4 (7,10,13,16), and 22:5 (4,7,10,13,16). Products of the linolenate series (the m3 family) include not only docosahexaenoic acid [22:6 (4,7,10,13,16,19)] but 20:s

140

KENNETH J. LONGMUIR

(5,8,11,14,17)and 225 (7,10,13,16,19)as well. Some polyunsaturated fatty acids of the oleate family [e.g., 22:4 (4,7,10,13)] are also found. The reader is referred to the reviews of Holub and Kuksis (1978) and Kuksis (1978) and to the data tables of White (1973) for thorough compilations of the fatty acid profiles of the various phospholipid classes, the various subcellular membranes, and the various mammalian tissues.

D. Asymmetry of Phospholipids in Subcellular Membranes

The concepts of lipid asymmetry in membranes have been thoroughly reviewed (Op den Kamp, 1979; Etemadi, 1980; Krebs, 1982). These reviews discuss the evidence for the asymmetric distribution of lipids and offer critical appraisals of the methods used for determining lipid asymmetry. The absolute asymmetry found for membrane proteins is not found for membrane lipids. Lipids of all classes can be found on both sides of a membrane. The best data for preferential enrichment of lipid on one side of a membrane are for the erythrocyte. This asymmetry was first demonstrated by chemical labeling studies, when it was discovered that phosphatidylethanolamine and phosphatidylserine are preferentially distributed on the intracellular (cytoplasmic) side of the erythrocyte membrane (Bretscher, 1972a,b; Gordesky and Marinetti, 1973). Studies with phospholipases and sphingomyelinase confirmed the enrichment of phosphatidylethanolamine and phosphatidylserine on the intracellular side and indicated that phosphatidylcholine and sphingomyelin are preferentially distributed on the extracellular side (Verkleij et al., 1973; Kahlenberg et al., Renooij el al., 1976). These results have been confirmed recently by the use of nonspecific phospholipid transfer proteins (Crain and Zilversmit, 1980). Lipid asymmetry is less well established for intracellular membranes. However, consistent data have accumulated for the mitochondrial inner membrane (Crain and Marinetti, 1979; Krebs et al., 1979; Harb et a / . , 1981). These studies have used ( I ) chemical probes for labeling the free amino group of phosphatidylethanolamine, (2) phospholipases, and (3) antibodies to cardiolipin. The results of these experiments indicate that phosphatidylcholine is somewhat enriched on the cytoplasmic side of the inner mitochondrial membrane, whereas phosphatidylethanolamine and cardiolipin are located mostly on the matrix side. An asymmetric distribution of lipids across microsomal membranes has not been conclusively demonstrated, even though microsomal preparations can be obtained which are clearly asymmetric with respect to marker enzymes. The lack of conclusive data may be due to the inherent difficulties

BIOSYNTHESIS AND DISTRIBUTION

OF

LIPIDS

141

with the methods used to demonstrate asymmetry. The impermeant reagents needed to modify one side of the membrane may in fact cross to the other side. Alternatively, the lipids of the endoplasmic reticulum may undergo flip-flop across the membrane during the time course of the experiments used to determine asymmetry. Lipids are synthesized on the cytoplasmic face of the endoplasmic reticulum (Section IV,G), yet newly formed lipid must accumulate on both sides ofthe membrane. Hence, flipflop of lipid from the cytoplasmic side to the lumenal side seems physiologically reasonable. Evidence for rapid movement of lipid across microsomal membranes is supported by "P-NMR experiments (de Kruijff et ul., 1978) which suggest that a portion of the microsomal lipid exhibits isotropic motion on the time scale of the N M R experiment. Such N M R data are consistent with the concept that a small portion of the membrane lipid exists in an arrangement other than a lipid bilayer, where rapid motion in all directions can take place to redistribute lipid from one side to the other. These observations suggest that asymmetry of the endoplasmic reticulum in vivo (and microsomal membranes in vifro)as a static phenomenon may be irrelevant. 111.

DESATURATION AND ELONGATION OF FATTY ACIDS

The principal product of dr mvo fatty acid biosynthesis in mammalian tissue is palmitic acid (16:O). De niwo fatty acid biosynthesis takes place in the cytoplasm in a cycle of reactions catalyzed by the multifunctional fatty acid synthetase complex. The reactions leading to the formation of fatty acid from acetyl-CoA and malonyl-CoA are well understood, and the regulation of the fatty acid synthetase complex has been recently reviewed (Wakil el d.,1983). Fatty acids can be modified ( I ) by desaturation reactions which introduce cis double bonds and (2) by elongation reactions which lengthen the fatty acid from the carboxyl end by two carbons per step. Desaturation of fatty acids in mammalian liver tissue can occur at positions nine, six, five, and four from the carboxyl end. These reactions are catalyzed by separate desaturase enzymes, termed the A", Ah, A', and A4 desaturases (James, 1977). Mammalian cells do not possess desaturase activity which will introduce a double bond beyond position 9 from the carboxyl group. Fatty acids with cis double bonds beyond position 9 are obtained by elongation of existing unsaturated fatty acid or by dietary uptake. Linoleic [ 18:2 (9,12)] and linolenic [18:3 (9,12,15)] are the two principal fatty acids supplied by dietary uptake and are called essential fatty acids. They are required for synthesis of polyunsaturated fatty acids and for prostaglandin formation.

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KENNETH J. LONGMUIR

Fatty acid produced by the fatty acid synthetase complex and fatty acid taken up from the circulation must be converted to the fatty acyl-CoA thioester by the enzyme acyl-CoA synthetase before elongation and desaturation can occur. Desaturase enzymes are membrane bound and are located in the microsomal fraction of the cell. The A9 desaturase system (also called the stearoyl-CoA desaturase) is a complex of three proteins: (1) NADH-cytochrome b, reductase, (2) cytochrome b5,and ( 3 ) the terminal desaturase enzyme, often called the cyanide-sensitive protein. The NADH provides reducing power to perform the oxygen-dependent introduction of the double bond, > 1. While sufficient to create ordered arrays of bilayer membranes, the energy barriers effected by van der Waals attraction are weak in any practical mechanical sense. It has become possible in recent years to test the DLVO tbeory experimentally by measuring force as a function of distance at 1 A resolution (Rand, 1981; Israelachvili, 1982). These measurements have shown a remarkable verification of the theory for mica surfaces in dilute electrolyte solution. However, there are many systems for which the DLVO theory clearly fails, the most notable example for our interests being interactions between bilayers. LeNeveu et al. (1976) measured the work of removal of water from an ordered array of phospholipid bilayers and used X-ray diffraction to observe the structural consequences of water removal. Short-range repulsive forces became evident from the fact that bilayers made from zwitterionic, electrically neutral, phospholipids repel each other in aqueous solution at separations up to 30 A. With decreasing separation the force grows exponentially with a characteristic decay length of 2.5-3.0 A. As bilayers approach contact, repulsive pressures can grow to the order of I000 atmospheres. These short-range forces have been attributed to solvation effects at surfaces, which give rise to a repulsive “hydration” force. Figure 2 is a linear plot of bilayer interaction energies in the vicinity of energy minima with and without the addition of charged lipids, The exponentially dropping hydration repulsion either is followed by a more gradual electrostatic repulsion followed by van der Waals attraction, or, for uncharged surfaces, it is balanced at shorter distance by the van der Waals attraction. Gruen et af. (1984) have developed a theoretical framework to explain the exponentially decaying hydration repulsion forces. According to their model, changes in the structure of water near an interface are associated with a nonzero value of an order parameter P, the dielectric polarization of water. It is represented as a vector with a zero value in bulk water and a maximal value in a state where water molecules are fully oriented in one direction (see Fig. 3). Although the decay of the polarizing perturbation reflects the properties of the aqueous medium, the value of P depends on properties of the membrane surface. Thus the importance of head group packing density becomes clear: zwitterionic compact surfaces, which have their charges near each other, will cause a weak polarizing perturbation. This is borne out by the data on differences in interaction energies between bilayers made of PC and PE (see Fig. 2). [For details see references in Parsegian and Rand (19831.1 The polar groups of bulky liquid-chain PC molecules pack poorly and must be surrounded with polarized water, whereas PE forms tightly packed bilayers. Figure 2 shows that the equilibrium distance for egg PE is about 10 A closer to the surface than that

229

MEMBRANE FUSION

A

Separation

20

I

40

(A) 60

80

I

-.03 0

Separation 20

0

-.2

I

I

40

(A) 60 1

'

3

_ - - -_ _ _ - - - -

I

FIci. 2. Linear plots of bilayer interaction energies in the vicinity of energy minima. ( A ) Difference in depth and location of energy niinima with and without the presence of electrostatic interactions. ( B ) Difference in interaction energies of egg PC and egg PE as a function of distance. (From Parsegian and Rand. 1983. with permission.)

for egg PC, and that the energy minimum is about 10-fold as deep. Also consistent with this model is the reduced hydration repulsion of PC in the gel phase, which is tightly packed, and therefore expected to polarize less water, as compared with PC in the fluid phase (Parsegian and Rand, 1983). Since hydration poses a formidable barrier to fusion, it follows that fusion of lipid bilayers will be facilitated by treatments or agents which cause dehydration of the surface. Bilayer surfaces are dehydrated by addition of polyethylene glycol, formation of a dehydrated calcium-phos-

230

ROBERT BLUMENTHAL

Membrane

I

/ J

W\J\

FIG.3. Polarization of water near a surface. The arrows represent the dielectric polarization of water. The maximal value is fully oriented perpendicular to the surface. I t decays as a function of distance from the surface.

phatidylserine (Ca-PS) complex, or freezing and thawing (Hui et al., 1981; Strauss, 1984). As we shall see in Section VIII,D dehydration is not sufficient to induce membrane fusion; such treatments also cause destabilization of the bilayer. The dependence of vesicle-vesicle fusion on the phospholipid head group is also consistent with their relative hydration energies. Duzgunes et al. (1981a) found that inclusion of PE in PS vesicles greatly enhanced divalent cation-induced fusion whereas inclusion of PC was inhibitory. Sundler and Papahadjopoulos ( 198 1) compared divalent cation-induced fusion rates of vesicles with phospholipids containing different (negatively charged) head groups-. They found that PA was most fusogenic, followed by PS, and no fusion with PI, consistent with the bulkiness and hydration energies of the head groups (Loosley-Millman et al., 1982). Similar results where obtained with pH-dependent fusion induced by VSV G protein reconstituted in lipid vesicles (Eidelman et d.,1984). B. Membrane Electrostatics

From the discussion in the previous section it is clear that the depth of the energy minimum is considerably decreased, and the equilibrium distance is considerably increased, when the membrane surfaces are charged. [For a calculation of the electrostatic energy between two charged spheres (vesicles) see Gingell and Ginsberg (1978) or Ohki et ul. (19821.1 Therefore, it is reasonable to expect charge neutralization to be a necessary condition for aggregationlfusion. This has been borne out experimentally. The presence of fixed charges on a membrane surface gives rise to a surface potential, 9. Free cations from the medium accumulate in the interfacial region and screen the charges. The distribution of these cations can be described approximately by the classical Gouy-Chapman formulation for the diffuse double layer. A balance is struck between electrostatic attraction of the cations to the negative charges at the membrane surface

231

MEMBRANE FUSION

and the entropic tendency of the cations to spread uniformly throughout the medium. At physiological ionic strength. the surface potential falls off nearly exponentially from the membrane surface with a characteristic distance of 9 A. The surface potential can be related to the surface charge density by an expression derived by Grahame (1947). Divalent cations are more effective at screening than are monovalent cations, but calculations indicate that screening is unlikely to be a major factor for charge neutralization by divalent cations, which bind more strongly to bilayers made of negatively charged lipids than to those made of zwitterionic lipids. The effects of this binding on )I can be calculated from a Stern equation, which is a combination of the Langmuir isotherm for surface adsorption of each ion, a Boltzmann expression for each ion, and the Grahame expression for surface charge density [see McLaughlin et (11. (1981) and Weinstein et nl. (1982) for more details]. Calculation of surface potentials requires data on binding constants of ions to phospholipid membranes. The binding of Ca’+ to PS membranes has been measured in a variety of ways including estimation of the effect of Ca” on monolayer and bilayer potentials, N M R relaxation, equilibrium dialysis, use ofCa’+sensitive electrodes, and 6 potential measurements. If the right corrections are made for the effect of screening, as predicted from diffuse double layer theory, and for the effect of monovalent cation binding (Na’), then the data in the literature are consistent with binding constant of 12 M ’ for the Ca-PS complex (McLaughlin et [ I / . , 1981). Using this value, McLaughlin ef a / . (1981) showed that the Stern equation is capable of accurately describing 5 potential data for PS vesicles and mixed PS/PC vesicles in 0. I M NaCl for a variety of concentrations of divalent cations. Thus, in theory, charge neutralization for mixed PS/PC vesicles can be determined for any given ionic condition. However, this does not work out experimentally. PS vesicles aggregate and fuse in about I niM Ca’ + and 100 mM Na’. According to McLaughlin r t rrl. (1981) the surface potential is about -40 mV under those conditions, and one would not expect those vesicles to aggregate and fuse. The discrepancy is due to the fact that it has been assumed that there is one affinity and a 1: I stoichiometry for the Ca-PS complex. However, under conditions in which membranes aggregate there is a dramatic discontinuity in the binding curve with a change to a 1.2 stoichiometry for the complex (Ekerdt and Papahadjopoulos, 1982). The discontinuity has been shown with pure PS. PS/PE ( I : 1 ), and PS/galactosylcerebroside (10: I ) . but not with PS/PC I: I . The higher affinity 1 : 2 complex is interpreted as the formation of a new type of cooperative binding site (possibly the anhydrous complex) between closely apposed bilayer membranes. X-Ray diffraction (Portis et (11.. 1979). N M R (Hauser et a/.. 19771, and freeze-fracture data (Papahadjopoulos r t

+

232

ROBERT BLUMENTHAL

al., 1975) also point to the existence of such a complex. The difference between PS/PC and PS/PE in their ability to form such a complex is consistent with their relative hydration energies (see Section VIILA). The equilibrium distance for PC-containing membranes is large enough to inhibit interbilayer molecular contacts, even after complete neutralization of the surface charge at higher concentrations of Ca”, which decreases JI sufficiently to allow formation of stable aggregates. Aggregation seems to be well described by the charge neutralization model, whereas for fusion a superthreshold amount of divalent cation binding is required (Duzgunes et al., 1981b). For instance, aggregation of pure PS vesicles can be induced by high concentrations (0.55 M ) of Na’ alone (Day et al., 1980). The equilibrium distance for this aggregation process is larger that for Ca2+-inducedaggregation. The process is very different from the Ca-PS complex formation between membranes, and there is no significant bilayer destabilization, which is apparently required for fusion, in this case (see Section VIIi,D). [For a more detailed review on cation binding, aggregation, and fusion, see Nir et al. (19831.1 C. Membrane Deformation

In the previous section I noted that stable contact between two surfaces will be achieved if G >> kT. With interaction energies at the energy minima of -0.01 erg/cm2 for PC and 0.1 erglcm’ for PE (Parsegian and Rand, 1983), a contact between two single apposing phospholipid head groups (area 70 A) will yield energies of 0.002 and 0.02 kT units, respectively (kT = 4 X erg). However, a contact between 100 phospholipid head groups will increase these energies 100-fold. Therefore, spherical vesicles need to be deformed to provide this area of contact. This deformation induces a tension on the membrane, and work has to be performed to deform against this countervailing tension. A contact angle 0 is produced that obeys the relationship (Parsegian and Rand, 1983). cos e

=

I

+ c/2T

(5)

where G is the interaction energy per unit area, and T the membrane tension. Rigid spheres have a very small area of contact, but flaccid vesicles can be deformed to form large contact areas before stresses build up (Evans and Parsegian, 1983). The contact area is in fact ITR’ sin’ 8, where R is the radius of the vesicle and 0 the contact angle (see Fig. 4). For a contact angle of 15” and R = 100 A the energy gained from contact (for PE) is 0.05 kT units; for a larger vesicle of 1000 A radius, this energy becomes 5 kT units. This simple calculation shows that for the same contact angle a small vesicle will form a smaller area of contact and therefore a less

233

MEMBRANE FUSION

FIG.4. Two mutually deforming spherical vesicles of radius R . Tis the membrane tension and H (he contact angle.

stable contact. However, as mentioned earlier the behavior of SUVs is opposite to that of LUVs. In the fluid phase SUVs are rigid, in contrast to LUVs which are readily deformed in that phase. SUVs will more readily aggregate and fuse than large vesicles, not because of their ability to form large areas of contact but because of their susceptibility to destabilization (see Section VIILD). In considering the energetics for contact formation we must consider the energy of deformation of vesicles. According to Evans and Parsegian ( 1983) the strength of contact vis-a-vis thermal or mechanical perturbations is represented by -yA,

+

W , - AFI-R >> kT

(6)

where y is the free energy reduction per unit area of contact formation. A, the area of contact, W , the work of deformation, which includes deformation of the membrane and of interior contents, and AF,., i s the integrated long range attraction between the undeformed particles. The contact angle can be calculated by minimizing Eq. (6) with respect to 0. The deformation will give rise to stretching of the bilayer, because the round shape has the lowest surface area to volume ratio. If the volume does not change, a change in shape will lead to an increase in surface area

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ROBERT BLUMENTHAL

and consequently the membrane will be under stress. Beyond a 3% increase in area, the bilayer will burst (Evans and Skalak, 1979). If the energy minimum is attained with a contact angle consistent with an area increase of less than 3%, a stable aggregate is formed. Such aggregates are probably formed with PS vesicles in high salt. On the other hand, if the energy minimum is beyond the 3% area increase a defect will be formed that might result in fusion or lysis. A simple calculation shows that a 3% area increase will be attained at a contact angle of about 50”. If the membrane is highly permeable to water (“leaky”), the new volume will readjust without tension on the surface and stable contacts will be formed without destabilization of the membrane. Consequently there will be no fusion. Since membrane leakiness is not well defined. the rate of water permeation must be considered relative to the rate of deformation. D. Membrane Destabilization

From the preceding discussions it is clear that charge neutralization and removal of water from the surface (dehydration) are necessary, but not sufficient, conditions for lipid bilayer fusion. Charge neutralization of certain lipid mixtures by certain mono- or divalent cations can give rise to aggregation but not fusion (see Section VII1,B). Dehydration by addition of sucrose, dextran, or glycerol does not cause membrane fusion (Boni er a / . , 1984). Dehydration by freeze-thawing can be protected against by substances such as glycerol, DMSO, ethylene glycol, and sucrose. These substances are cryprotective because they prevent formation of large ice crystals, which presumably cause the defects in membranes. The crucial event in lipid bilayer fusion seems to be membrane destabilization or the formation of defects in the lipid bilayer.” I have to be somewhat unclear about the nature of these defects, since their molecular arrangements are not well defined. However, it is reasonable to assume that in such defects the nonpolar fatty acyl chains will be transiently exposed to water. This can then give rise to hydrophobic attraction between two membranes which have such defects. Israelachvilli and Pashley (1982) have measured the attractive forces in aqueous solution between two mica surfaces that were rendered hydrophobic by adsorption of a monolayer of the cationic surfactant hexadecyltrimethylammonium. They found an exponentially decaying hydrophobic force in the same range of separation distance as the van der Waals force but with an energy of attraction about an order of magnitude higher. This hydrophobic force is not observed in ’An alternative mechanism, i.e.. formation of inverted micelle intermediates, will be discussed in Section IX.

MEMBRANE FUSION

235

intact lipid bilayers. where it seems to be “neutralized” by the local structure of water molecules interacting with phospholipid head groups. According to Ohki ( 1984) the “hydrophobicity” of the membrane surface is related to the surface free energy measured in a phospholipid monolayer. The surface tension of a monolayer, consisting of negatively charged lipids, increases in the presence of divalent cations. The concentration of divalent cations required t o produce this effect corresponds to the threshold concentration required to induce vesicle fusion. Other factors, such a s the degree of vesicle curvature, temperature. and membrane expansion by osmotic swelling (see below), all exhibit a similar correlation between increased surface energy and tendency of the membranes to fuse. On the basis of these results Ohki proposed that the increased hydrophobicity of the membrane surface is responsible for membrane fusion. Hui pf rrl. (1981) have observed by freeze-fracture electron microscopy “point defects” in bilayers containing a mixture of egg PC and soybean PE, which were fused by freeze-thawing. They appeared as “rivets” between adjacent bilayers in cross-fractured areas. Although those structures are usually highly transient, they seem to have been preserved in the multilamellar samples containing highly unsaturated PC and PE. As mentioned in the previous section, bilayer defects can be formed in Ca”-induced lateral phase separation, which results in the formation of rigid crystalline domains of acidic phospholipids within a mixed lipid membrane (Papahadjopoulos, 1978). Polyethylene glycol (PEG) can induce such defects along domain boundaries by binding to the phospholipid bilayer and inducing local rigidification of the bilayer (Boni rt l l / . , 1984). In cells Honda et ( I / . (1981) found that certain components contained in a commercial grade PEG are fusogenic for cells and that recrystallized PEG is nonfusogenic. “’ However, recrystallized PEG is fusogenic for lipid vesicles (Boni ct nl., 1984). Presumably liposomes are destabilized by PEG alone, whereas cell membranes are more resistant to destabilization and need an additional perturbant. Osmotic swelling is also required for PEGinduced cell fusion (see below) but not for PEG-induced liposome fusion. As mentioned before, cis-unsaturated fatty acids at high concentrations can render membranes fusogenic by creating ”defects.” Lyso-PC, which has been shown to be a fusogen in a number of systems (Lucy, 197x1, probably also works according to that mechanism. The notion that destabilization is a crucial factor in bilayer fusion is “‘Keci-ystallized PEG did induce cell fusion in the assay of Smith r / t i / . (19x2). On the other hand. Wqjcieszyn 1’1 ( I / . (19x3) found a difference in the effects of nonfusogenic and fusogenic PEG on cellh. Presumably the polymer itself promotes dehydration and clow apposition of adjacent cell membranes. but fusion requires the destabilization provided hy the additives contained in commercial PEG.

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ROBERT BLUMENTHAL

also consistent with the observation that small vesicles, which have a high degree of curvature, have an intrinsically greater capacity to fuse than larger vesicles (Liao and Prestegard, 1979; Wilschut et al., 1980; Nir et al., 1982; Boni et al., 1984; Ohki, 1984; Morris et al., 1984). The strain imposed on the strongly curved bilayer of an SUV (Mason and Huang, 1978) makes it highly susceptible to destabilization. I have noted in a preceding section that SUVs made of PC with saturated chains will spontaneously fuse below T, (the liquid phase transition temperature). SUVs can only partially freeze because of molecular packing restrictions, and consequently “defects” in the boundaries between solid and liquid domains will be created. Presence of bilayer defects are indicated by leakage of internal contents of liposomes during fusion. Morris et al. (1985) found that SUVs made of PS/PE rapidly lose their contents during Ca*’-induced fusion, consistent with their susceptibility to destabilization, whereas large vesicles with the same lipid composition are nonleaky during the first few cycles of fusion. However, the extent of leakage through the defects is dependent on a number of factors, such as how transient the defects are, how large they are, and how closely apposed the membranes are. Therefore, leakage might or might not be observed in a fusion event mediated by defects. Formation of gross defects in membranes by high-intensity electric field pulses has proved to be a very effective means to induce membrane fusion (Zimmermann and Vienken, 1982). Membrane contact between at least two cells is achieved by “dielectrophoresis” generated by an alternating nonuniform electric field, while reversible electric breakdown in the membrane contact zone is achieved by application of a field pulse of high intensity. This technique has promising biotechnological applications. In the previous section I noted that membrane deformation induced by apposition could lead to destabilization. However, membrane stretching induced by osmotic swelling is an even more powerful driving force for membrane fusion. The intracellular or intravesicular volume expansion gives rise to the membrane tension (stretch). Defects will be formed that might result in fusion when the membrane area increases to about 3% (see Section IX,C). In their studies of liposome-planar bilayer fusion, Akabas et al. (1984) have clearly shown that osmotic swelling of liposomes, which are tightly bound to the planar bilayer in a “prefusion” state, is an essential condition for fusion of vesicular and planar membranes. Osmotic swelling is also a necessary condition for PEG-induced fusion of cells. The PEG induces close apposition of the cell membranes (“prefusion” state), but fusion requires osmotic swelling brought about by removal of PEG or by dilution (Knutton, 1979a; Wojcieszyn et al., 1983).

MEMBRANE FUSION

237

Experiments showing that Ca”- or ATP-evoked catecholamine release from isolated chromaffin granules can be inhibited by increasing the osmotic strength of the medium led to the suggestion that osmotic swelling might be an important step in granule-plasma membrane fusion during exocytosis (Edwards et u / . , 1974; Pollard r t d . , 1976). The fact that hyperosmotic solutions inhibit exocytosis in intact secretory cells (see references in Pollard et NI.. 1982, and in Akabas et NI., 1984) and in the cortical granule preparation (Zimmerberg, 1984) lends support to this idea. Moreover, in mast cells secretory vesicle swelling concomitant with ex1984). Although osocytosis has been visualized directly (Curran (’[ d., motic swelling seems to be required for fusion in these systems, it is still unclear what the mechanisms for generating the osmotic force are (Baker and Knight, 1984). E. Bilayer Fusion Mediated by Soluble Peptides or Proteins

1 have summarized in Table I1 a number of polymers, peptides, or proteins that have been reported to induce vesicle-vesicle fusion. This list is most probably incomplete, and 1 apologize if I have left out anyone’s favorite fusion protein. These soluble proteins are often regarded as models for natural fusion proteins. The list reveals two basic requirements for the ability of the protein to mediate fusion: cross-linking and destabilization of bilayers. In almost all cases shown in Table 11, there is also leakage of liposome contents, although not necessarily with the same time course or at the same protein concentration as required for fusion. With S U V s . protein-induced destabilization alone is in some instances sufficient to induce bilayer fusion. For example, uncharged PC vesicles are not likely to be cross-linked by agents such as alamethicin, melittin, or bovine serum albumin. As I noted before, S U V s in the solid phase are inherently unstable, and they will fuse by themselves due to interactions between hydrophobic edges (see Section VII1,D). Similarly, the proteins or peptides in Table I1 may create such edges in fluid phase SUVs, and thereby induce fusion. The interaction of (basic) proteins or peptides with acidic liposomes results in fusion by cross-linking as well as by destabilization. This has been illustrated by Eytan and Almary (1983), who compared the eficiency of various cations to induce fusion in PC/PE/CL vesicles. The efficiency was in the following order: Ca” < La” < polylysine < polymyxin B = melittin. Both polymyxin B and melittin are amphipathic molecules with a hydrophobic moiety and a hydrophilic one bearing 5 positive charges. Since it has been shown that at higher concentrations these molecules will lyse cells and disrupt membranes, it is reasonable to assume

TABLE 11 SOLUBLE PEP~IDES AND PROTEINS THATINDUCE BILAYER FUSION (Po1y)peptide Alamethicin Melittin Bovine serum albumin Albumin fragment Concanavalin A Tubulin Clathrin Myelin basic protein Polylysine, cytochrome c Polylysine, cytochrome c Polyhistidine Polymyxin B Melittin Polyamines Synexin

Liposome type"

suv suv suv

suv suv

suv suv

suv SUV SUV

suv suv

suv

LUV LUV

Liposome compositionh PC PC PC PC

Pc PC PC PCIP a PE PCIPEIPPCIPEIPPCIPEIP PCIPEIPPCIPEIPPE/P~

Reference Lau and Chan (1973) Morgan et a/.(1983) Schenkman er a/.(1981) Garcia et a/.(1984) van der Bosch and McConnell (1975) Kumar et a / . (1982) Blumenthal e? a / . (1983) Lampe and Nelsestuen (1982) Stollery and Vail (1977) Gad et a/.(1982) Wang and Huang (1984) Gad and Eytan (1983) Eytan and Almary (1983) Schuber et a/.(1983) Hong er a/.(1982)

SUV, Small unilamellar vesicle, LUV, large unilamellar vesicle. PC. Phosphatidylcholine; P - , acidic phospholipids (PA, PS, or CL): PA. phosphatidic acid; PS, phosphatidylserine; CL. cardiolipin; PE. phosphatidylethanolaine. "

MEMBRANE FUSION

239

that the hydrophobic tail will insert into the bilayer and destabilize it while the charged moiety will serve as the cross-linker. Some proteins or peptides listed in Table I 1 will not destabilize the bilayer by themselves. Therefore, additional factors arc required. such as the use of solid phase S U V s (myelin basic protein and concanavalin A), passage through the phase transition (melittin-PC and concanavalin A), incubation at elevated temperature (alamcthicin). addition of lyso-PC (myelin basic protein), high amounts of PE in the liposome (polylysine, polyhistidine, polymyxin B, melittin, polyamines, synexin), lowering the pH (polyhistine, clathrin, bovine serum albumin), and addition of niillimolar divalent ions (tubulin). The majority of proteins shown in Table I1 use S U V s , which are easier to destabilize, as targets for fusion. Exceptions are synexin (Hong (/I., 1982) and polyamines (Schuber P / ( i / . , 1983). which mediate fusion of large vesicles. These molecules presumably enhance aggregation, while fusion is induced by interaction of divalent cations with the lipid bilayer, causing phase separation and destabilization, according to Papahadjopoulos ( 1978). However, in liposomes containing high mole fractions of PE, spermine and spermidine induced fusion in the absence of divalent cations. In this case the lipid head group provided the destabilization necessary for fusion. Fusion mediated by these soluble proteins helps sharpen our concepts concerning mechanisms of membrane fusion mediated by natural fusion proteins. For example. based on the three-dimensional structure of the ectodomain of the HA glycoprotein of influenza virus (see Section V ) , and on physicochemical studies of pH-dependent conformational changes of that domain (Skehel (/I., 1987), the following picture has emerged for pH-dependent fusion mediated by influenza virus (see White c/ (/I., 1983): The protein is a trimeric rod-shaped molecule 135 A in length, consisting of a stem and three globular. highly folded domains (stalks) at the top. The globular domains are composed of the HA1 chain, and, a s they contain the binding sites for sialic acid. they provide the cross-linking feature. The stem contains the hydrophobic HA2 N-terminal sequences implicated in fusion (fusion peptide). As the pH is lowered, the stalks open up and expose the fusion peptide which then interacts with the target membrane, destabilizes it, and thereby induces fusion. Although this seems to be an attractive scheme, one of its main problems is that the stalk reaches about 100 A out of the viral membrane, and consequently the fusion peptide has to move up all that way to bind to and destabilize the target membrane. Perhaps structural and physicochemical studies on viral fusion proteins reconstituted into lipid bilayers (see Section IX,D) might yield further insights into their mode of action.

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IX. MEMBRANE FUSION: FACT, HYPOTHESIS, OR THEORY? A. Introduction

The field of membrane fusion has been plagued by a great deal of confusion and controversy as to both what is the correct theory and what are the facts. Therefore it might be worthwhile to examine this problem according to the “Scientific Method.” The facts consist of what we observe, and the theories consist of what we suppose or invent. The suppositions are made sometimes to fill the gaps in our factual knowledge, sometimes to increase the understanding of facts, and sometimes for other reasons. For instance, the theory of evolution is not an account of a process which anyone has observed from beginning to end. It is a supposition, albeit not a complete supposition for it incorporates some observed facts. It has been invented to bring some sense, some intelligibility, into what otherwise would have been brute senseless facts. The “Scientific Method” is a logical sequence of steps followed in any scientific research activity of observing, forming hypotheses, experimenting, testing hypothesis, and controlling or predicting subsequent action on the basis of test results. In doing so we construct models which we can manipulate and which allow us to interpret and test hypotheses against reality. Through tests, analyses, and modifications we construct an acceptable model. The final structure and logic of the model are the basis for predictions that allow solution of the problem. In the literature on the philosophy of science there is some diversity in views as to what constitutes a “hypothesis” or “model” or “structure” on the one hand, and a “theory” on the other hand. I consider an “hypothesis” to be a result of observation and inference, followed by some deductive testing of the conclusions. A “theory,” on the other hand, is the conclusion of a thorough analysis of observations and inferences, examination of the deductive tests, invention, and analysis and testing of multiple hypotheses. Finally, it is a conclusion that has gained acceptance by the scientific community and withstood the test of time. B. Artificial Lipid Membranes

What makes lipid bilayers come close together or what holds them apart can at this point be fitted into the framework of a theory. The foundations of the theory are based on the chemistry and physics of surfaces and on the theory of colloid stability. Both areas have been studied intensively during the past century. Part of the problem can be described according to DLVO, and this has been supplemented in the past 17 years by con-

24 1

MEMBRANE FUSION

sideration of the short-range repulsive forces. The nature of these forces has been established by the highly accurate measurements and theoretical insights of the Bethesda-St. Catherine group (Parsegian, Rand, and company) and the Canberra school (Israelachvilli, Pashley, Marcjela, Grucn, and company). However. what happens after the membranes come together is still unclear. Figure 5 shows two possible intermediate structures in fusion of lipid bilayers. The inverted micelle has been proposed by a number of authors as a possible intermediate (Neher, 1974; Lau and Chan, 197.5; Pinto da Silva and Nogueira, 1977; Cullis and Hope, 1978; Rand, 1981). This inverted micelle is related to the H I , phase of phospholipids and is detected as a lipidic particle by freeze-cleavage (Verkleij and Ververgaert, 1978) and by "P-NMR (Cullis and de Kruijff, 1978). The inverted micelle is likely to exist as an intrvbilayer rather than as an inrvtrbilayer intermediate (see footnote 4). Cullis and Hope (1978) observed that certain agents (fusogens), which induce fusion between erythrocyte membranes, also induce the Hi, phase in a portion of the isolated erythrocyte membrane. Certain cone-shaped phospholipids such as PE and CL-Ca' ' favor the formation of the Hi, phase. Verklcij r ? 611. (1979) showed that C a L t induced fusion of vesicles composed of egg PC/CL ( I :I ) was associated with formation of lipidic particles. On the other hand, with the same lipid

Pentalarninar

It Defect A H u i et al., 1981)

Fused

FIG 5

Two model5 for intermediate\ in membrane fuqion. See Section 1X.B for diwuswm.

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mixture Bearer et ul. (1982) observed no formation of lipidic particles during fusion events arrested by quick freezing in the absence of glycerol as a cryoprotectant. Siegel (1984) pointed out that it would be very hard to detect intermediate structures in vesicle-vesicle fusion even by quick freezing, since they have very short half-lives. Moreover, the lowering of the temperature required for the freeze-cleavage might perturb the intermediate structures, which are temperature sensitive. The other possible intermediate, shown in Fig. 5, is a “defect.” Formation of such “defects” can be induced by a variety of mechanisms, discussed in Section VII1,D. The nature of the defect might remain quite elusive since it is transient and its possible irregular structure is not amenable to techniques, such as X-ray diffraction, designed to resolve structures. They have been observed by Hui et 01. (1981) in mixtures of egg PC and soybean PE that which were fused by freeze-thawing. In the resulting multilayers, numerous lipidic particles were observed by freezefracture electron microscopy. They appeared as “rivets” between adjacent bilayers in cross-fractured areas. Since both the defect (Hui et al., 1981) and the inverted micelle (Cullis and de Kruijff. 1978) display an isotropic 3’P-NMRsignal superimposed on the broad bilayer signal, the NMR technique will not distinguish between the two structures. Moreover, the size of the defect should be about equal to that of the inverted micelle intermediate, which is about 12.5 nm in diameter (Siegel, 1984). It is therefore hard to decide on morphological grounds whether the “rivets” observed by Hui et al. (1981) are defects rather than inverted micelle intermediates. Since defects as well as inverted micelle intermediates have both concave and convex surfaces, similar cone-shaped lipids will favor their formation. Since the intermediate might be either a defect or an inverted micelle depending on the experimental conditions (lipids, temperature, the way fusion is induced), it is not necessary to postulate either one as part of a general mechanism of membrane fusion. Perhaps more insight will be gained into the nature of the intermediates or types of intermediates required through development of sophisticated biophysical techniques (NMR, fluorescence) to monitor local perturbations, which are a very small fraction of the overall global structure. In addition, theoretical insight into the nature, energetics, and probability of such intermediates might be gained from molecular dynamics modeling of the bilayer structure. C. Natural Membranes

Fusion of natural membranes is still in the realm of hypothesis. Many of the factors, such as charge neutralization and dehydration, that are

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required to make lipid bilayers come together and fuse do not pertain to natural membranes. The areas of contact between membranes are usually small in viral fusion and exocytosis. Moreover we are still unclear about the facts concerning biological membrane fusion, specifically whether it occurs in “bare lipid patches,” or via proteinaceous structures (see the discussion in Section 11). The studies of the early stages of exocytosis captured by rapid-freezing of Litnrrlirs amebocytes (Ornberg and Reese, 1981) make an argument for the latter proposition. Within seconds of stimulation, the plasmalemma buckles inward to form broad appositions with secretory granules lying near the cell surface. The distribution of intramembrane particles is not different from that over the rest of the plasmalemma. Numerous punctate pentalaminar contacts form between granular and plasma membranes. and an exocytotic opening begins at a minute pore at one of these contacts. The pore then progressively widens as the granule contents expand and diffuse into the space around the amebocyte. The capacitance patch clamp studies of mast cell degrdnuhtion support the notion of an exocytotic pore. Fernandez and Neher (1984) captured single exocytotic events using this technique and observed rapid increases and decreases (”tlickering”) of the capacitance steps, consistent with the opening and closing of an “exocytotic” pore. The hypothesis of a pore or junction structure in the fusion event is attractive since in general proteins rather than lipids provide regulation of biological events. However, in viral fusion it has been shown in a number of studies that presence of protein in the target menibrane was not required (White and Helenius, 1980: Maeda ct ( I / . , 1981; White ct u / . , 1982; Hsu et ( i / . , 1983; Haywood and Boyer, 1984). Although the viral membrane obviously contains the regulatory protein(s) required for t’usion. the possibility of forming a junctional complex between proteins in hotli membranes becomes less likely. Therefore we must leave open the qucstions of the reality of a junctional complex or of the universality of biological fusion mechanisms. D. Toward Reconstitution of Membrane Fusion A possible critique (as 1 noted in Section 11) of the use of liposomes to model membrane fusion is that artificial bilayer fusion might be completely different from natural membrane fusion. So why use model systems? A valuable lesson may be learned from the study of ionic channels. Extensive studies on model channels (such as gramicidin and EIM) in bilayers had been carried out long before single channel behavior had been observed in natural membranes. Many of the techniques and the concepts of the mechanism of ion permeation through channels (reviewed by Blumenthul

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and Klausner, 1982), and the nature of opening and closing of voltagedependent channels (Ehrenstein et al., 1974), had been developed. Thus the foundations had been laid to study the problem of ionic channels in biological systems. Similarly, a conceptual framework for approaching the problem of membrane fusion is being developed with these model systems. Since proteins are one way or another involved in natural membrane fusion, a happy medium between artificial and natural fusion is proteinmediated fusion. Early attempts to model protein-mediated involved the effects of (fusion) irrelevant proteins (see Section VII1,E). However, the most promising avenue is reconstitution of proteins that have been destined by nature to fuse. Reconstitution has proven to be a very effective way to study the mode of action of transport proteins and of membrane receptors in a well-defined environment (Klausner ef al., 1984). A reconstitution of a fusion protein in a planar bilayer has been reported by Zimmerberg et ul. ( 1980b). A water-insoluble membrane-associated protein with a high affinity for Ca” was isolated from calf brain synaptosomes and inserted into a planar bilayer. Fusion of liposomes with this reconstituted bilayer could then be induced at 10 p M Ca”. The Ca”-binding protein has not been characterized structurally, and its role in promoting fusion in vivo is not known. On the other hand, viral spike proteins have been very well characterized both structurally and functionally (see Section V). X-Ray crystallography and studies of pH-dependent conformational changes have yielded some insights into their mode of action (see Section VII1,E). They have been reconstituted into lipid vesicles by detergent dialysis. Reconstituted Sendai virus envelopes have been used extensively to introduce macromolecules into animal cells and to implant membrane proteins (receptors, transport proteins) into plasma membranes of living cells (reviewed by Loyter and Volsky, 1982). Reconstitution requires solubilization of a membrane protein in an appropriate detergent in the presence of lipid followed by removal of detergent by dialysis or other means. Removal of detergent results in formation of closed vesicles. The nature of the detergent, the molar ratio of protein, lipid, and detergent, and the mode of detergent removal are all very important factors in successful reconstitution (Klausner et ul., 1984). Use of detergents with a low critical micelle concentration (cmc) such as Triton X-100 have not been as effective in reconstitution studies as detergents with a high cmc, such as deoxycholate and octylglucoside. Reconstitution studies with Sendai virus have predominantly been carried out with Triton X-100, since other detergents such as deoxycholate fail to solubilize the relevant spike glycoproteins. Loyter and co-workers (see Loyter and Volsky, 1982) have very successfully injected macromolecules into cells and implanted membrane proteins into plasma membranes, using Sendai envelopes reconstituted from Triton X-100. However,

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those systems suffer from similar problems as encountered with liposomecell interaction in that it is not clear what components of cell-associated Sendai envelopes are due to fusion, endocytosis, stable adsorption, or exchange (see Section 111). On the other hand, the efficiency of the transfer process is much higher than that for pure liposomes." In order to study mechanisms reconstituted systems that are better defined physicochemically need to be developed. One such system has recently been developed by Eidelman et NI. (1984). Purified G protein of VSV was reconstituted in egg PC vesicles by detergent dialysis of octylglucoside. Conditions for obtaining a homogeneous population of fusion-competent reconstituted vesicles, as shown by electron microscopy and fluorescence energy transfer, include a high protein to lipid ratio and a slow removal of the detergent. The fusion activity is nonleaky and is dependent on pH, temperature, and presence of negatively charged lipid such as PA or PS in the target membrane. The system is still imperfect, since its pH dependence does not match the biological pHdependence and fusion with biological membranes is not as efficient as with liposomes as targets (0.Eidelman and R . Blumenthal. unpublished observations). However, with the improvement of reconstitution techniques and with appropriate target membranes, the possibility of functional reconstitution that matches the biological situation is very close.

ACKNO W L E D 6 M ENTS

I thank Drs. Sen-Wek Hui and Jacob Israelachvili for helpful dibcussions. and Drs. Ofer Eidelman, Stephen Moms, Richard Omberg. Adrian Parsegian, Harvey Pollard, David Siegel. Anne Walter. and Joshua Zimmerberg for their careful consideration of the mnnuscript and useful comments. The reference list was compiled with the aid of MEDLINE and completed in December. 1984.

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LeNeveu. D. M.. Rand, P. M., and Parsegian. V. A. (1976). Measurement offorces between lecithin bilayers. Ntifirre (London) 259, 601-603. Liao, M.-J., and Prestegard, J. H. (1979). Fusion of phosphatidic acid phosphatidylcholine mixed lipid vesicles. Biochirn. Biopliys. Acfci 550, 157-173. Llinas. R. R., and Heuser, J . E., (eds.) (1977). Depolarization-release coupling systems in neurons. Neiirosci. Res. Proguu~nBirll. 15. Loosley-Millman. M.. Rand. R. P., and Parsegian. V. A. (1982). Effects of monovalent ion binding and screening on measured electrostatic forces between charged phospholipid bilayers. Biophys. J . 40, 22 1-232. Loyter. A,. and Volsky, D. J . (1982). Reconstituted Sendai virus envelopes as carriers for the introduction of biological Material into animal cells. Cell Sirrf. Rev. 8, 215-266. Lucy, J. A. (1978). Mechanism of chemically induced fusion. Cell Srwf Rev. 5 , 267-304. McLaughlin. S., Mulrine, N . , Gresalfi. T., Viao, G . ,and McLaughlin, A. (1981). Adsorption of divalent cations to bilayer membranes containing phosphatidylserine. J . Gen. PIiysiol. 77, 445-473. Maeda, T., Kawasaki. K.. and Ohnishi. S.-I. (1981). Interaction of influenza virus hemagglutinin with target membrane lipids is a key step in virus-induced hemolysis and fusion at pH 5.2. Proc. N m l . A c ~ d S. c i . U.S.A. 78, 4133-4137. Markwell, M . A . K.,Svennerhold. L..andPaulson,J.C. (1981). Specificgangliosidesfunction as host-cell receptors for Sendai virus. Puoc. Narl. A c d . Sci. U.S.A. 78, 5406-5410. Margolis. L . B. (1984). Cell interaction with model membranes. Probing, modification and simulation of cell surface functions. Biochim. Biophvs. Acfci 779, 161-189. Mason, J . T.. and Huang, C. (1978). Hydrodynamic analysis of egg phosphatidylcholine vesicles. Ann. N . Y . Accrd. Sci. 308, 2 9 4 9 . Mayhew, E.. Papahadjopoulos. D., Rustum. Y . M., and Dave. C. (1976). Inhibitionof tumor cell growth in vi/ro and in viw by I-o-arabinofuranosylcytosineentrapped within phospholipid vesicles. Cancer. Res. 36, 4406-441 I . Mazurek, N . , Schindler, H., Schiirholz, T.. and Pecht, I . (1984). The cromolyn binding protein constitutes the Ca” channel of basophils opening upon immunological stimulus. Proc. N o / / . Actid. Sci. U . S . A . 81, 6841-6845. Morgan. C. G . . Williamson, H.. Fuller, S . , and Hudson, B. (1983). Melittin induces fusion of unilamellar phospholipid vesicles. Biochirn. Biophys. Actri 732, 668-674. Morris, S . J . . and Hughes, J . M. X . (1979). Synexin protein is non-selective in its ability to increase Ca’ +-dependentaggregation of biological and artificial membranes. Biocllern. Biopliys. Rc,.s. Comnrirn. 91, 345-350. Morris, S. J . , Chiu. V. C. K . , and Haynes, D. H. (1979). Divalent cation-induced aggregation of chromaffin granule membranes. Memhr. Biocliem. 2, 163-201. Morris, S . J . . Costello, M. J.. Robertson. J . D..Sudhof. T. C., Odenwald. W. F.. and Haynes. D. H. (1983). The chromaffin granule as a model for membrane fusion implications for exocylosis. J . Aicfon. Nerv. S y s f . 7, 19-33. Morris, S . J.. Gibson, C. C . . Smith. P. D., Greif, P. C., Stirk, C. W., Bradley, D., Haynes, D. M., and Blumenthal, R. (1985). Rapid kinetics of Ca”-induced fusion of phosphatidylserine-phosphatidylethanol vesicles. J . B i d . Clrew~.260, 4122-4127. Mukherjee, B., Orloff, S . . De Butler, J.. Triche, T., Lalley, P., and Schulman, J . D. (1978). Entrapment of metaphase chromosomes into phospholipid vesicles (lipo-chromosomes): Carrier potential in gene transfer. Proc.. Ncirl. Actid. S c i . U.S.A. 75, 1361-1365. Neher, E. (1974). Asymmetric membranes resulting from the fusion of two black lipid bilayers. B i o ~ h i / ? rBiophys. . AC/O373, 327-336. Neher. E., and Marly, A. (1982). Discrete changes of cell membrane capacitance observed under conditions of enhanced secretion in bovine adrenal chromaffin cells. Proc.. Nrrrl. A c u ~ S. C ~U.S.A. . 79, 67124716.

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25 1

Nigg. E. A , , Cherry. R. J.. and Bachi. 7’. (1980). Influence of influenza and Sendai virus on the rotational mobility of hand 3 proteins in human erythrocyte membranes. Viroloqv

107. 552-556. Nir, S.. and HentL, J . (1978). On the forces between phospholipid bilayers. J . Coil. / t i / c r : / ; Sci. 65, 399-4 14. Nir. S.. Wilschut. J.. and Hentz. J. (1982).Thc rate o f fusion of phospholipid vesicles and the role of hilayer curvature. B i ( ~ h i / i iBiopliy.s. . Ac,rir 688. 27s-278. Hentz. J.. Wilschut. J.. and Duzgunes. N . (1983). Aggregation and fusion o f phoslipid vesicles. Pro,y. Sur Nihhizuka, Y . (19x4). Turnover

holipids and signal trancduction. Sc.icnt.c

225, 1365-1370. Ohki. S. ( 1984). Effects of divalent cation\. tcniperatui-e, osmotic pressure gradient. and vesicle curvature on phosphatidylserine vesicle fusion. J . Mcr77br..Biol. 77, 265-275. Ohki. S..Duzgunes. N., and I,eonards. K . (1982). Phospholipid vesicle aggregation: Effect of monovalent and divalent ions. Bioc~Iictni.\/ry21, 2 127-2 133. Orci, I>.. and Perrelet. A . (1978). Ultrastructurul aspects of exocytotic membrane fusion. Ccll S/fr:/: ROI’. 5 , 629-656. Ornherg. K. L.. and Keese. 7’. S . (1981). Beginning ofexocytosis captured by rapid-freeAng of /.;iirrr/rt,\ amebocytes. J . C d l Riol. 90, 40-54. Ostro. M., Giacomoni. D.. Lavelle. I>.. Piixton. W., and Dray. S.(1978). Evidence for the translation of rabbit globin niKNA after liposome mediated insertion into a human ccll line. Ntrrurc (Loritloti) 274, 92 1-923. I’agano. K. E . . and Weinstein. J. N. (1978). lntefiictions ofliposonies with mammalian cells. .4unrf. Rcl.. Biopl1y.c. Biocr7g. 7, 435-463. Palade, G. (1975). Intracellular aspects o f the process o f protein synthesis. S(,it,ric t’ 189,

347-358. Palade. Ci. E.. and Bruns. R. R. ( 1908). Structural modulations of plasmalemmal vesicles. J . CP// Biol. 37, 633-649. Papahad.jopoulos. D. ( 1978). Calcium-induced phase changes and fusion in natural and model membranes. Cell Sw/: Rci.. 5, 765-7YO. Papahadjopoulos. D.. Vail. W. J.. Jacohson. K . . and I’oste. G . (1975). Cochleatc lipid cylinders: Formation hy fusion of unilaniellar lipid vesicles. Bioc,lrirn. Biopliys. At.rri 394,

483-49 I. Papahadjopoulos, D., Wilson. T., and Taber. K. (19x0). Liposonies a s vehicles for cellular incorporation of biologically active maci-oiiiolcculr~./ r r Virro 16, 49-54. Parsegian. V . A,. and Rand. K. 1’. ( 19x3). Membrane interaction and deformation. Sur-fxc forces in biological systems. Arrti. N . Y . Ac t r d . S(.i. 416, 1-12. Pastan. 1. H., and Willingham. M. C. (19x1 ). Jour-ney to the center of the cell: Role of the 214, 504509. rcceptosome. Scierrc~c~ Pastcrnak. C. A , . and Micklem. K . J . (1974). The biochemistry of virus-induced cell fusion. Changes i n membrane integrity. Bioc~/ic,rii.J . 140, 405-41 I . Pearse. H . M . F . . and Bretcher. M . S. (19XI). Membr;ine recycling by coated vesicles. Arirrrt, Rcv. Rifdfc’rr7. 50. 85-101, Pinto da Silva. P.. and Hranton, D.(1970). Membrane splitting in freere etching. Covalently hound ferritin as a membrane marker. J . C’c// / j i o / . 45, 598-605. Pinto da Silva. P.. and Nogueira. M . L. (1977). Membfiine fusion during secretion. A hyzoospores pot hesis based on electron microscope observation of /’/iytop/rrhortr ptr/r~ii~ortr during encystment. J . Cell Biol. 73. Ihl-1x1. Pollard. H. H . . Zinder. 0 . .Hoffman. 0. G . . and Nikode-icvic. 0 . (1976). Regulation of the transmembrane potential of isolated chromafiin granules by ATP. ATP analogs and external pH, J . B i d . Clic~rii.251, 4544-4550.

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Pollard. H. B., Creutz, C. E., Fowler, V . , Scott, J . , and Pazoles, C. J . (1982). Calcium-dependent regulation of chromaftin granule movement, membrane contact, and fusion during exocytosis. Cold Spring Harbor Symp. Quant. Biol. 46, 819-834. Portis, A,, Newton, C., Pangborn, W., and Papahadjopoulos, D. (1979). Studies on the mechanism of membrane fusion: Evidence for an intermembrane Ca2+ phospholipid complex synergism with MgLt and inhibition by spectrin. Biochemistry 18, 780-790. Poste, G . , and Nicholson, G . C. (eds.) (1978). Membrane fusion Cell Surf. R e v . 5. Poste, G . , and Papahadjopoulos, D. (1976). Lipid vesicles as carriers for introducing materials into cultured cells. Influence of vesicle lipid composition on mechanisms of vesicle incorporation into cells. Proc. N u l l . Acad. Sci. U . S . A . 73, 1603-1607. Poste. G.. and Pasternak, C. A. (1978). Virus induced cell fusion. Cell Surf. R e v . 5 , 305367. Rand, R. (1981). Interacting phospholipid bilayers: Measured forces and induced structural changes. Annu. R e v . Biophys. Bioeng. 10, 277-3 14. Rink, T. J., Sanchez, A., and Hallan, T. J . (1983). Diacylglycerol and phorbol ester stimulate secretion without raising cytoplasmic free calcium in human platelets. Nature (London) 305, 317-319. Robertson, J. D. (1959). The ultrastructure of cell membranes and their derivatives. Biocltem. Soc. S y m p . 16, 3-43. Rosenberg, J., Duzgunes, N., and Kayalar, C. (1983). Comparison of two liposome fusion assays monitoring the intermixing of aqueous contents and of membrane components. Biochim. Biophys. Acta 735, 173-180. Satir. B. (1974). Ultrastructural aspects of membrane fusion. J. Supramol. Strucrr. 2, 529537. Schenkman, S., Araujo, P. S. , Dijkman, R., Quina, F. H., and Chaimovich, H. (1981). Effects of temperature and lipid composition on the serum albumin-induced aggregation and fusion of small unilamellar vesicles. Biochim. Biophys. Acia 649, 633-641. Schlegel, R.. and Wade, M. (1984). A synthetic peptide corresponding to the NH2 terminus of vesicular stomatiti's virus glycoprotein is a pH-independent hemolysin. J. B i d . Chem. 259, 4691-4694. Schlegel. R., Willingham, M. C.. and Pastan, I. H. (1982). Saturable binding. Saturable binding sites for vesicular stomatitis virus on the surface of Vero cells. J . Virol. 43, 871-875. Schlegel, R., Tralka, T. S., Willingham, M. C., and Pastan, I. (1983). Inhibition of VSV binding and infectivity by phosphatidylserine: Is phosphatidylserine a VSV-binding site'? Cell 32, 639-646. Schneider, A. A., Cline, H. T., Rosenheck, K., and Sonenberg, M. (1981). Stimulus secretion coupling in isolated adrenal chromaffin cells: Calcium channel activation and possible role of cytoskeletal elements. J. Neurochern. 37, 567-575. Schuber, F., Hong, K., Duzgunes, N.. and Papahadjopoulos. D. (1983). Polyamines as modulators of membrane fusion: Aggregation and fusion of liposomes. Biochemistry 22, 6 134-6 140. Schullery, S . E., Schmidt, C. F., Felsner. P., Tillack, T. W., and Thompqon. T. E. (1980). Fusion of dipalmitoylphosphatidylcholinevesicles. Biochemistry 19, 3919-3923. Siegel, D. P. (1984). Inverted micellar structures in bilayer membranes. Formation and half-lives. Biopkys. J. 45, 399-420. Silvius, J. R., and Gagne, J. (1984). Calcium effects on lipid lateral distribution and fusion in mixtures of synthetic phosphatidylserines, phosphatidylcholines and phosphatidylethanolamines. Biopphys. J . 45, 169a. Siraganian, R. P.. Urata, C., and McGivney, A. (1983). Arachidonic acid release during IgE

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and Ca”4onophore activation of rat basophilic leukemia cells. Monogr. Allergv 18, 120-123. Skehel, J., Bayley, P., Brown, E., Martin, S. . Waterfield. M., White, J.. Wilson. I . , and Wiley. D. (1982). Changes in conformation of influenza virus hemagglutinin at the pH optimum of virus-mediated membrane fusion. Pro0.5 pm in diameter), such as a bacterium or another cell. While characteristic of “professional” phagocytic leukocytes (neutrophils, macrophages), this form d endocytosis occurs in most cells. Pinocytosis, on the other hand, involves the formation of much smaller vesicles (0.1-0.2 pm) resulting in the internalization of extracellular fluid and dissolved solutes (“fluid phase pinocytosis”) as well as any macromolecules bound to the plasma membrane at the site of vesicle formation (“adsorptive endocytosis”). The internalization of macromolecules bound to specific cell surface receptors is referred to as receptormediated endocytosis. In many cells, all pinocytosis is initiated by the invagination and subsequent vesiculation of clathrin-coated pits at the plasma membrane (Marsh and Helenius, 1980; Steinman et a l . , 1983). Apart from the sizes of the vesicles involved, the actual differences between pinocytosis and phagocytosis are not clear. While the formation of large ( > I km) phagocytic vesicles can be blocked by microfilamentdisrupting agents such as cytochalasin D, pinocytosis and the phagocytosis of smaller particles are generally unaffected (Steinman et al., 1983). Both types of endocytosis typically result in the delivery of internalized contents to hydrolase-rich lysosomes for digestion, and both are inhibited at low temperature (4°C). In addition, at least some types of phagocytosis may be associated with the formation of clathrin coats on the cytoplasmic surface of the nascent phagocytic vacuole (Aggeler and Werb, 1982; Aggeler et al., 1985; Montesano et al.. 1983). One important difference between the two processes, however, involves their regulation. Phagocytosis is a highly regulated event in mammalian cells: a phagocytic vacuole forms and is custom-fit in direct response to a particle binding to the appropriate receptors on the cell surface. In contrast, pinocytosis is generally thought to be a constitutive process, whereby pinocytic vesicles form continuously without the need for external signals. Pinocytosis can thus be likened to a continuously operating escalator, whereas phagocytosis behaves more in the manner of an elevator called into service only when needed.

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B. Membrane Recycling 1. QUANTITATIVE CONSIDERATIONS

As mentioned above, endocytosis can involve the internalization of enormous quantities of membrane. During one round of phagocytosis, a cell such as a macrophage can internalize as phagocytic vesicles an amount of plasma membrane equivalent to 50% of its initial surface area (Werb and Cohn, 1972; Steinman et ul., 1983; Petty et ul., 1981). During pinocytosis, typical mammalian cells in culture will continuously internalize 50-200% of their plasma membrane surface area every hour (Steinman et al., 1976). Nevertheless, a cell’s size and surface area are maintained at relatively constant values. Since the amounts of membrane involved are far in excess of a cell’s biosynthetic capacity, and since most cell surface glycoproteins are relatively long Lived ( t , , , 10-20 hr), it has long been presumed that internalized membrane components must be returned intact to the cell surface, i.e.. recycled (Steinman et ul., 1976; Silverstein et NI., 1977). Indeed, during the last several years a considerable body of direct and indirect evidence has been obtained for the existence of extensive and rapid membrane recycling during endocytosis (for review, see Steinman et ul., 1983; also, see below).

2. SORTING OF ENDOCYTIC VESICLEMEMBRANEA N D CONTENTS Since membrane recycling must occur via vesicular transport, a major conceptual problem is apparent: How can the continuous bidirectional vesicular traffic occur simultaneously with the continuous accumulation and/or degradation of internalized macromolecules in lysosomes? In fact, the intracellular accumulation of fluid-dissolved solutes is not quite unidirectional. Up to one-third may be lost from the cells back to the extracellular medium in the first few minutes following internalization. an expected consequence of vesicular recycling (Besterman et ul., 198 I ; Adams et ul., 1982; Swanson et u l . , 1985). Nevertheless, it is clear that there is a net accumulation of endocytosed solutes, indicating that at some stage after uptake there must be a sorting of endocytic vesicle membrane from contents: the membrane container returns to the cell surface while internalized solutes remain inside the cell. To a first approximation, this sorting event may simply reflect the geometry of endocytic and recycling vesicles: internalization in vesicles of low surface to volume ratio, recycling of membrane (and proportionately less fluid) in vesicles of relatively high surface to volume ratio. While the recycling vesicles have yet to be definitively identified, initial morphological evidence suggests that they may consist of small diameter (50 nm) vesicles

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or tubules (van Deurs and Nilausen, 1982; Geuze et uf., 1983; Beguinot et uf., 19841, structures having a much higher surface to volume ratio than the large, spherical coated vesicles (usually 0.2 Fm in diameter) which serve as the primary endocytic compartment. The available evidence strongly suggests that sorting of membrane from contents occurs soon after endocytosis, i.e., prior to the delivery of internalized tracers to lysosomes. As mentioned above, loss of endocytosed solutes due to membrane recycling is significant only during the first few minutes after uptake. Following delivery to lysosomes, there is relatively little reflux out to the extracellular medium (Besterman et af., 1981; Steinman et al., 1983). Similarly, in spite of extensive endocytosis and recycling, lysosomal enzymes-endogenous markers of lysosomal contents-are not lost at appreciable rates (Gonzalez-Noriega et al., 1980; Steinman et al., 1983). Recent work on receptor-mediated endocytosis has defined the prelysosomal site of sorting and recycling as a class of uncoated vesicles and tubules referred to as endosomes (Helenius et af., 1983), organelles whose properties will be considered in detail below. 111.

RECEPTOR-MEDIATED ENDOCYTOSIS

A. Receptor Recycling

The central importance of the endocytic pathway has been established by numerous findings over the last several years demonstrating that a variety of extracellular macromolecules bind to specific plasma membrane receptors and are internalized by endocytosis. Approximately 50 such examples have now been described, including receptor-mediated uptake systems for cell nutrients [e.g., cholesterol via low density lipoprotein (LDL), iron via transferrin, cobalamin via transcobalamin 111, polypeptide hormones [e.g., insulin, epidermal growth factor (EGF)], antibodies, lysosomal enzymes, modified plasma glycoproteins, bacterial toxins, and RNA and DNA viruses (e.g., influenza virus, poliovirus) (Steinman ef al., 1983). The existence of cell surface receptors greatly enhances the efficiency of endocytosis of specific macromolecules both by dramatically increasing their concentration at the plasma membrane and by targeting their uptake to selected cell types. The efficiency y is enhanced still further by the fact that receptors can be reutilized during ligand uptake. In many cases, the number of ligand molecules taken up per hour greatly exceeds the number of available receptors, even in the absence of new synthesis (i.e., cycloheximide-treated cells). Kinetic data show that an individual receptor can mediate the uptake of up to 10-20 ligand molecules every hour and

259

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therefore hundreds during the course of its lifetime (Steinman et al., 1983). Accordingly, it is clear that after internalization, receptors must recycle back to the plasma membrane. 6. Pathway of Receptor-Mediated Endocytosis

A general consensus has been reached concerning the basic features of the intracellular pathway taken by receptors and their ligands (for review, see Helenius et al.. 1983; Brown et al., 1983; Hopkins and Trowbridge, 1983). As diagramed in Fig. I , ligands first bind to their receptors, which results in the accumulation of the receptor-ligand complex at clathrincoated pits. Most investigators believe that the coated pits then pinch off to form coated vesicles (see Pastan and Willingham. 1983, for a different view), which rapidly lose their coats and fuse with endosomes, a heterogeneous population of vesicles and tubules in the peripheral and perinuclear cytoplasm. Due to their slightly acidic internal pH, endosomes

4

recycling vesicles pinocytic vesicles

endosomes

FIG. I ,

The basic pathway of receptormediated cndocytosis.

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facilitate the dissociation of many receptor-ligand complexes, thus allowing the return of free receptors to the cell surface and the transfer of the discharged ligand, now free in the endosome’s lumen, to lysosomes for degradation. This scheme (Fig, 1) clearly illustrates the role of endosomes as the central sorting station in the endocytic pathway, providing an intracellular site for receptor-ligand dissociation and recycling without requiring transit through the proteolytically perilous lysosomal environment. It also implies that acidic intraendosomal pH is crucial to maintaining the orderly traffic of membrane, receptors, and ligands in to and out from the cytosol. Having summarized the basic aspects of receptor-mediated endocytosis, in the next section we shall consider what is known (or suspected) concerning the mechanisms that control each step of the pathway. IV. MECHANISMS AND FUNCTIONS OF ENDOCYTIC ORGANELLES A. Coated Pits and Coated Vesicles 1. STRUCTURE AND ASSEMBLY

The composition and structure of the polyhedral lattice comprising the cytoplasmic coat of coated pits and coated vesicles has been well studied (for review, see Pearse and Bretscher, 1981; Harrison and Kirchhausen, 1983). The coat is composed primarily of the 180-kDa protein clathrin and its two associated light chains of 30-36 kDa. Clathrin normally exists as a trimer which takes the shape of a triskelion (Ungewickell and Branton, 1981; Kirschhausen and Harrison, 1981).Three light chains are associated with the clathrin heavy chains at the vertex of each triskelion. Several other nonclathrin proteins are associated with the coat including one or more 100-kDa species, tubulin, and a SO-kDa phosphoprotein (Pfeffer et al., 1983). The most important known property of clathrin coats is their capacity for self-assembly. Given the appropriate conditions of pH and ionic strength, clathrin triskelions will spontaneously form baskets, with or without an enclosed membrane vesicle. Conceivably, the favorable free energy change associated with the self-assembly process provides the driving force for membrane vesiculation, initiating the formation of an endocytic vesicle. Only clathrin and clathrin light chains are needed for assembly; thus, the role of the other associated coat proteins is unclear, although there is some suggestion that one or more 100-kDa species is required for attachment of the lattice to the membrane (Unanue ef al., 1981).

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261

In intact cells, the factors which regulate when and precisely where clathrin [or the 100-kDa protein(s)] attaches to the plasma membrane are unknown (see also Section IV,B). There is definite specificity to the attachment process, since not all organelles can serve as substrates for coated pit formation. Similarly, iri vitro, certain membranes (e.g., the erythrocyte membrane) are known not to permit clathrin binding and assembly (Unanue e t a / . , 1981). Large plaques of assembled clathrin in hexagonal arrays are often found at sites of focal adhesion to substrates or to other cells (Aggeler and Werb, 1982);whether adhesion signals clathrin assembly or simply prevents the subsequent vesiculation event is unclear. The only known inhibitor of coated pit formation (and the subsequent formation of coated vesicles) is depletion of cytoplasmic K’ concentrations (Larkin et al., 1983; Moya et a / . , 1985).The depolymerized clathrin presumably enters the preexisting pool of soluble clathrin (Goud et a / . , 1985) from which it can be recruited when K’ is returned to normal levels. Why K’ concentrations should exert this effect, however, is unknown.

2.

FORMATION AND UNCOAI’ING OF COATED VESICLES

The progressive transformation of a coated pit to a spherical coated vesicle is associated with the insertion of pentameric faces of triskelions into the planar hexagonal lattice (Heuser and Evans, 1980). While the addition of pentons serves to increase the curvature of the structure, pentamer formation may represent a cause or an effect. Whatever triggers the coated pit to coated vesicle conversion, it is clear that coated vesicles begin to lose their coats only moments after they form. Uncoating is presumably important not only to allow the coated vesicle to fuse with its intracellular target(s), but also to permit the reutilization of clathrin. Uncoating is unlikely to be spontaneous since the assembly process appears to be energetically favorable. Thus, it is of particular interest that the cytoplasms of most cells contain an ATP-dependent uncoating activity (Schmid et a / . , 1984; Braell rt al., 1984; Rothman and Schmid, 1986).The “uncoating ATPase” is a 70-kDa protein which binds both ATP and assembled clathrin heavy and light chains (Schmid et al., 1984). ATP hydrolysis is coupled to depolymerization of the clathrin basket.

B. Localization of Receptors at Coated Pits Since coated pits represent regions of the plasma membrane with a high probability of internalization, another manifestation of the efficiency of receptor-mediated endocytosis is the ability of receptors and receptorligand complexes to accumulate at coated pits. Some receptors, such as

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the LDL receptor, appear to be localized at coated pits even in the absence of bound ligand; others only after ligand attachment (Steinman et al., 1983). Thus, the signal for accumulation at coated pits cannot be ligand binding per se. Similarly, ligand binding cannot be the unique signal for the de n o w formation of a coated pit beneath the receptor. In this regard it is important to point out that coated pits are relatively catholic: a single coated pit can accumulate and mediate the internalization of several distinct receptors (Anderson et al., 1982). One possible mechanism for the localization of receptors at coated pits postulates a specific and conserved recognition event between the cytoplasmic domain of a receptor and some coat component (Pearse and Bretscher, 1981). While attractive, the nature of this recognition system is not apparent when one considers the structures of a variety of receptors. As illustrated in Fig. 2, “coated pit receptors” differ greatly in their overall

N

I I

COOH I

I

I 5

COOH

N

27aa

COOH

COOH

LDL

_-__*--

IU~W~LUI-

transferrin

EGF

_____ --recepror

_____I__

rscspror

COOH

N

N

C ASGP

Fc

...._ & ..

recepror

recepror

*..

insulin

VSV G

rscepror

prorein

-.

FIG.2.- Comparison of plasma membrane receptors that localize at coated pits. Each of the schematically diagrammed receptors mediate the uptake of their respective ligands via coated pits and coated vesicles. Only in the case of the LDL receptor is it clear that the receptor localizes preferentially at coated regions of the plasma membrane in the absence of bound ligand. N and COOH refer to amino- and carboxyl-termini, respectively. The sizes of the cytoplasmic domains (where known) are indicated in number of amino acid residues (aa). The overall molecular weights for each receptor are as follows: LDL receptor (160K). transferrin receptor (2 x YOK, disulfide-linked), EGF receptor (170K), asialoglycoprotein receptor (ASGP) (-54K). Fc receptor (55-60K). insulin receptor (2 x 90K. 2 x 125K. disulfide-linked tetramer), VSV G protein (5OK). For references, see Brown et rrl. (1983, review), Yamamoto et ul. (1984). Ullrich et ul. (1984, 1985). Chiacchia and Drickamer (19841, Green ef ril. (1985), Lewis e t a / . (1986), and Roth e t a / . (1986).

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sizes, in the sizes of their cytoplasmic tails, and in their orientations in the bilayer. Some receptors are phosphorylated or fatty acylated, some have multiple subunits joined by interchain disulfide bridges. A comparison of the receptors whose amino acid sequences are known reveals no unique primary structure homology (Brown et ul., 1983). Even several nonreceptor membrane proteins, such as vesicular stomatitis virus (VSV) G protein and leucine aminopeptidase, enter coated pits either constitutively or fol1986; Louvard, 1980; lowing the addition of specific antibody (Roth r t d.. H. Reggio and D. Louvard, personal communication). 1 . GENETIC EVIDENCE

Nevertheless, genetic evidence supports the importance of a membrane protein’s transmembrane and/or cytoplasmic domains in coated pit localization. The best example is provided by the human LDL receptor, a 160-kDa transmembrane glycoprotein with a single 50-amino acid cytoplasmic domain (Yamamoto et d . , 1984). Of the many mutations of the receptor known to disrupt receptor activity. one class blocks the efficient uptake of LDL by coding for a receptor protein that is unable to accumulate at coated pits (Goldstein et a / . , 1979). cDNAs corresponding to several mutant alleles have been cloned and sequenced, and in each case the defect was found to yield a receptor with an altered or truncated endodomain (Lehrman P I a l . , 1985; Russell et al., 1985). One mutation resulted from a single amino acid change (Tyr to Cys) at the eighteenth residue from the membrane’s cytoplasmic surface. In a second example, the transmembrane and cytoplasmic domains of VSV G protein were spliced onto the extracellular domain of the influenza virus spike glycoprotein, hemagglutinin ( H A ) , thus converting a protein which does not normally enter 1986). coated pits (the HA) to a chimeric protein which does (Roth et d., 2 . COMPOSITION OF COATED PITS

Given the great diversity in the structure of membrane proteins which enter coated pits, it is a priori unlikely that any recognition between a receptor and a coat component will turn out to be specific, in the sense of a ligand-receptor interaction. Instead, some aspect of a receptor’s oligomeric structure, which may be altered in response to ligand binding, rnay be a determining factor. For example, if receptors are simply aggregated by intermolecular interactions or by ligand binding, they rnay be relatively slow to diffuse through a coated region. Conceivably, the dense layer of coat proteins closely apposed to the cytoplasmic face of the membrane may physically impede the movement of a receptor’s endodomain. The slower rate of diffusion would increase the net residence time of a receptor at a coated pit, increasing the probability of internalization. Im-

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portantly, such a mechanism does not necessarily require the existence of specific interactions between receptors and coat components. Irrespective of the mechanism of receptor localization at coated pits, any interaction may not involve clathrin directly. Even a 50-amino acid long cytoplasmic tail is only 10-20 A long, and the clathrin basket may be as far as 150 A away from the membrane. Other coat proteins, such as the 100-kDa protein which is probably more closely apposed to the membrane than clathrin, may be more likely candidates. Clearly, coated pits generate distinct differentiated microdomains of the cell surface. At the very least, they differ biochemically from adjacent segments of the plasma membrane with respect to an increased concentration of receptors. Little quantitative or biochemical data concerning the concentration of other membrane proteins in coated pits have yet been obtained. However, one might expect that such nonreceptor proteins may be at least partially excluded from steric considerations alone: the accretion of receptor-ligand complexes may simply not leave much room. Exclusion is not likely to be 100% effective, i.e., coated pits probably are not perfect “sorters” which select out only receptors. All plasma membrane proteins exhibit finite turnover rates, and therefore are likely to be internalized and delivered to lysosomes (Steinman et al., 1983). In addition, mutant LDL receptors which fail to localize selectively at coated pits still mediate LDL uptake at a much slower rate (see Table 2 in Brown and Goldstein, 1976) that is comparable to the estimated rate at which plasma membrane surface area is internalized (see above): approximately one cell surface equivalent of bound LDL per hour. This would be the expected result if the mutant receptors were randomly distributed on the cell surface. Since coated pits occupy only I-2% of the surface area, and given the resolution of current morphological techniques, the detection of a randomly distributed components in coated pits would be difficult at best. C. Endosomes 1. DEFINITION OF ENDOSOMES

Following the uncoating of coated vesicles, the next station on the endocytic pathway is the endosome (for review, see Helenius et d.,1983). Although these structures were first described as intermediates on the pathway to lysosomes over 20 years ago (Straus, 1964), the extent of their importance in regulating membrane traffic during endocytosis has only recently been appreciated. Morphologically, endosomes are indistinct. They comprise a heterogeneous population of narrow tubules and vesicular elements distributed throughout the peripheral and perinuclear cytoplasm. Moreover, endosomes are not yet known to have any specific marker

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enzyme or other unique biochemical feature. The identification and definition of endosomes, therefore, is strictly functional: any uncoated vesicle which labels with endocytic tracers prior to their appearance in lysosomes. Endosomes in the cell periphery often seem to be labeled with internalized macromolecules prior to those in the perinuclear or lysosomal region (e.g., Wall er ul., 1980). Time-lapse photomicrographs also suggest that the peripheral endosomes actually migrate in toward the perinuclear region, perhaps along microtubule tracks, and perhaps fuse directly with lysosomes (e.g., Helenius et ul., 1983; Hirsch et ul., 1968; Herman and Albertini, 1984). The known functions of endosomes include the following (Helenius et ul., 1983): ( 1 ) dissociation of receptor-ligand complexes; (2) targeting of receptors back to the cell surface (or some other organelle); (3) targeting of dissociated ligands to lysosomes; (4) discharge of iron from internalized transferrin; ( 5 ) site of penetration of enveloped animal viruses (influenza virus, VSV, Semliki Forest virus) into the cytosol; and (6) site of penetration of many bacterial toxins, such as diptheria toxin, into the cytosol.

2. STRUCTURE OF ENDOSOMES By analyzing computer-generated three-dimensional reconstructions, we have found that “typical” endosomes consist of a large central vesicle (0.4-1 .O p,m in diameter) from which 2-10 narrow (