ANNUAL REVIEWS
Further
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ENZYME TOPOLOGY OF
+946
INTRACELLULAR MEMBRANES Joseph W. DePierre and Lars Ernster Department of Biochemistry, Arrhenius Laboratory, University of Stockholm, S-106 91 Stockholm, Sweden
CONTENTS PERSPECTIVES AND SUMMARY .................. ... ...................... ......... ................... ... INTRODUCTION ........... ......... . . . ........................................ ...... ........ .. ................
202 203
Contemporary Concepts of Membrane Structure ... ..... ....... . . .. ..... .. ......... Methodology for Studying the Topology of Membranes .. .. ................. . ..... Enzyme topology in the lateral plane ...... ........................ ............. ............ ........... Enzyme topology in the transverse plane . ............................... .................... ........ Lipid topology .... ............................. ...... ..................... .............. ............................ Other methods ................................................... ............................ .......................
204 205 205 206 206 207
MITOCHONDRIA ................................................................................................ ........
207
Morphology ............. . ............................................................................... .. .. ........ The Outer Membrane ................................. ................ ........ ................................. The Inner Membrane .............. ........... ...................... ................... .......................
207 207 209 209 210 212 214 214 215 217 218 220 221 222 226 227 229 230
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General features ...... ................... ......... ........................................... ..................... General aspects of lateral topology ...... .................. ............ .................................. General aspects of transverse topology .................... ............................................ The respiratory chain, ATPase, and related energy-transducing systems ...... ... General features................................................................................................ NADH-Q reductase (Camp/ex I) ... .. ... ........... ............ .................... ........... .... Succinate-Q reductase (Complex II)................................................................ Ubiquinone ... ..... ....... ..................... .................. ...... ............ .......... ..... ................. QHrcytochrome c reductase (Complex III) ................................................ ... Cytochrome c .................................................................. ..................... ............. Cytochrome c oxidase (Comp/ex IV) ................ .............................................. Transhydrogenase ..................... ... ......... ... ...................................... ...... ........ .... ATPase .............................................................................................................. Ion trans/ocators .. .. ................. ... ................ ... ........... ................ ... ........ ... ... ..... Other enzymes ......... .......... ..... ... ..................................... ......... .......... ...... ... ...... .... .
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DePIERRE & ERNSTER
CHLOROPLASTS ........................... ...............................................................................
General Aspects........................................ .............. ............ ...............................' " ... Photosystems I and II and Coupling Factor CF} ............................................ Localization 0/ Chlorophyll .................................................................................. Interactions between the Photosystems and Chlorophyll-Protein Complexes................................................................................................ Stacking 0/ Thylakoids..........................................................................................
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231 231 233 235 236 236
THE ENDOPLASMIC RETICULUM ........................................................................
237
General Aspects........................................................................................................ Topology in the Lateral Plane.............................................................................. Topology in the Transverse Plane........................................................................ Binding of Membrane Proteins............................................................................ Complexes? .............. . . . .. .. ... . ... .... . . . ...... . . .. . .. ... . . . .. . . .. . . .. ... .. ... .. . . . . . .. . . . . . . Relationship of Proteins to Lipids ...................................................................... Functional Consequences of Topology ................................................................
237
THE G OLGI APPARATUS..........................................................................................
246
General Aspects........................................................................................................ Topology in the Lateral Plane.............................................................................. Topology in the Transverse Plane........................................................................
247
THE NUCLEAR ENVELOPE .. . . . . . ...... . . . . . ....................................................................
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C ONCLUSION.. ........................................................... ........ . . .. . . . . . . . ................................
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PERSPECTIVES AND SUMMARY
The functional importance of the intracellular membranes of eukaryotic cells has long been apparent. The isolation and characterization of these structures have played a key role in the spectacular progress toward bridg ing the gap between cell and molecule that has taken place during the last 30 years. However, appreciation of the intimate relationship that exists between the function and topology of intracellular membranes is still grow ing. It is fascinating to see how, as the attention of more and more investiga tors is focused on this relationship, new techniques for studying membrane topology are developed, precise questions are formulated, and the answers begin to take shape. The basic structure of virtually all biological membranes is now thought to be the phospholipid bilayer. But how are proteins bound to this bilayer? How deep do they penetrate? Do they have special interactions with the phospholipid molecules immediately surrounding them? Are proteins dis tributed homogeneously (i.e. randomly) in the plane of the bilayer (the lateral plane) and across the membrane (the transverse plane)? If not, why not? Do proteins move in the lateral and transverse planes of the mem brane? If so, how rapidly? If not, why not? Many of these questions have been formulated as a result of studies on energy-transducing membranes, i.e. the inner mitochondrial membrane, the
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chloroplast thylakoid membrane, and the plasma membrane of prokaryotes. An important breakthrough in this field was the realization that the en zymes involved in electron transport and ATP synthesis are capable of giving rise to and interacting through electrochemical gradients across the membrane, gradients that constitute a functional link between these pro cesses and the process of active transport. While these discoveries give a solid physical framework to our understanding of cellular energy transduc tion, the underlying chemical mechanisms are still poorly understood. Con tinued progress in this field will be critically dependent on determining the reaction mechanisms of the catalysts involved and their topological rela tionship to one another in the membrane, as well as the dynamics of this topology. One of the most important results of studies on the membrane topology of the endoplasmic reticulum is a model for how certain integral proteins bind to their membranes. Cytochrome b5, NADH-cytochrome b5 reduc tase, and probably NADPH-cytochrome c reductase all have hydrophobic tails especially designed for anchoring them to the phospholipid bilayer. In addition, all enzymes whose distributions in the lateral and transverse planes of the endoplasmic reticulum have been carefully investigated have been found to be heterogeneously distributed. In most cases the structural and functional reasons for such distributions are not understood. Since the membrane of this organelle also contains two electron-transport chains (the cytochrome h5 system and the cytochrome P-450 system), evidence for the existence of macromolecular complexes has been much sought after; but to date, the evidence obtained cannot be unambiguously interpreted. Topological studies of the membrane of the Golgi apparatus and of the nuclear envelope have just begun, and the topologies of other organelles such as lysosomes and peroxisomes are still unknown. The future not only will hold further attempts to answer questions such as those formulated above but also will bring new questions about the details of the structure of the hydrophobic portion of the membrane, the dynamics of membrane topology, the interactions between different intracellular membrane sys tems, and the biogenesis of membranes. To answer them will require, as always, continued interest, ingenuity, and a little impatience. INTRODUCTION
Eukaryotic cells contain a variety of membranous organelles, at least some of which are probably the descendants of prokaryotic ancestors (1). The mitochondrion, the chloroplast, the endoplasmic reticulum, the Golgi ap paratus, the nucleus, the lysosome, and the peroxisome are examples of such
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organelles. Their membranes separate gene replication and transcription from gene translation, glycolysis from respiration and photosynthesis, in tracellular processes from the completion and transportation of products destined for secretion, etc. This compartmentation of cellular functions is of fundamental importance for the regulation of growth and differentiation and was presumably necessary for the evolution of multicellular organisms. As boundaries between different cellular compartments, the membranes of organelles are the sites where interactions between these compartments take place. A number of proteins, in particular transport (and "receptor"?) proteins, have evolved to control these interactions. In addition, intracellu lar membranes contain a variety of proteins with other functions as well, e.g. the electron-transport chains of the mitochondrion, chloroplast, and endoplasmic reticulum, and the enzymes of steroid and phospholipid me tabolism. In some cases the protein components of these metabolic path ways interact directly without mobile low-molecular-weight intermediates, and the membrane apparently maintains these components properly ori ented toward one another. In other cases the substrate of an enzyme may be part of the membrane itself or may "dissolve" in the bilayer; it is appar
ently efficient to have such an enzyme bound to the membrane. In still other instances, such as in energy-transducing membranes, enzymes may interact "chemiosmotically" by way of electrochemical gradients across the mem brane. By some as yet poorly understood process, biological membranes are assembled with a precise, asymmetric topology that is vital to the perfor mance of their cellular functions. This chapter is a survey of our current knowledge of the enzyme topology of intracellular membranes. To make it manageable, we have found it necessary to exclude the cell membrane [which has often been reviewed elsewhere (2-6)] and to exclude intracellular membranes of a rather special ized nature, such as synaptic vesicles, chromaffin granules, or the sarcoplas
mic reticulum. In addition, there is very little known about the enzyme topology of the membranes of a number of common organelles, such as lysosomes and peroxisomes. Here we discuss mitochondria, chloroplasts, the endoplasmic reticulum, the Golgi apparatus, and, very briefly, the nu clear envelope. We begin with a synopsis of current concepts of membrane structure and a survey of methods employed for the study of membrane topology. Contemporary Concepts of Membrane Structure
It is now generally accepted that most of the phospholipid in most biological membranes is arranged in a bilayer with the polar head groups at the two surfaces. A more controversial question is the extent to which this bilayer is interrupted by membrane proteins. These proteins are divided into two
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categories (7-13): peripheral (or extrinsic) proteins, which are thought to lie largely or completely on the aqueous side of the plane defined by the polar head groups of membrane phospholipids; and integral (or intrinsic) proteins, which are thought to pass through this plane and to make substan tial contact with the hydrocarbon region of the membrane. The bulk of the lipid in biological membranes seems to have no special structural relation ship to the membrane proteins (e.g. 9). However, results obtained with certain membrane proteins [for example, cytochrome c oxidase and rho dopsin (12)] suggest that they may have important structural interactions with the phospholipid molecules immediately surrounding them. It is now clear that most, if not all, peripheral and integral proteins are asymmetri cally distributed in the transverse plane of the membrane, and often in the lateral plane as well (3, 10, 1 4-16; see also below). Only recently has it become clear that different phospholipid species are also asymmetrically distributed in the transverse plane of the membrane (17-24). The structure of biological membranes is in a constant state of flux. Under physiological conditions most of the lipids diffuse rather freely in the plane of the membrane (13, 25-30) and their hydrocarbon chains are disor dered (10, 26, 27, 30-33). A number of membrane proteins also seem to diffuse rather rapidly in the lateral plane of the bilayer (34, 35), a finding that has given rise to the fluid mosaic model of membrane structure (9, II, 12, 36). One the other hand, transverse rotation of phospholipids ("flip flop") and of membrane proteins is apparently a very slow process (34, 35, 37, 38). Methodology for Studying the Topology of Membranes ENZYME TOPOLOGY IN THE LATERAL PLANE At present, the most widely used techniques for investigation of enzyme topology in the lateral plane of membranes are subfractionation (including disruption of the mem brane into smaller pieces) (6, 39-41), normal (39, 42) and freeze-fracture (10, 34, 43, 44) electron microscopy, and reconstitution of membranes from isolated components (39, 45, 46). Other techniques have been developed e.g. cross-linking of membrane proteins to one another (47-49) and photo lysis-induced cross-linking of the fatty acid moiety of phospholipids to pro teins in their immediate vicinity (50, 5 1 )-and should find broader application. The first of these two new methods should allow "nearest neighbor analysis" of membrane proteins, while the latter should allow investigation of possible specific associations between membrane proteins and the phospholipid molecules immediately surrounding them, as well as determination of which parts of a protein extend down into the hydrophobic region of the membrane.
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ENZYME TOPOLOGY IN THE TRANSVERSE PLANE Some of the most widely used approaches for determining the transverse distribution of mem brane proteins are listed in Table 1. As with all experimental techniques, these approaches involve hazards of which the investigator must be con stantly aware (2, 40). For instance, many of these methods utilize sub stances (proteases, lactoperoxidase, antibodies, small charged molecules) to which membranes are supposed to be impermeable. This impermeability should never be assumed, but should always be demonstrated. On the other hand, many of these approaches are reliable only when a positive result is obtained. For example, if a protease or antibody or nonpenetrating reagent fails to inhibit an enzyme present on the membrane of a closed vesicle, it must be shown that these agents do inhibit the enzyme when the vesicle is opened, before any conclusions can be drawn. LIPID TOPOLOGY The topology of membrane lipids is almost certainly related-biogenically and/or functionally-to the topology of membrane proteins. Table 2 lists some of the methods that have been used to study the transverse distribution of membrane lipids in biological membranes. Of these approaches, treatment with purified phospholipases has been the most widely used and is the only method whose application to intracellular membranes has been reported (79). This study revealed that phospholipid species are asymmetrically distributed in the transverse plane of the mem brane of microsomes, the inner mitochondrial membrane, the Golgi mem brane, and the lysosomal membrane (Figure 1). In the future, phospholipid exchange proteins will surely be more widely used in studies of phospholipid distribution in membranes. Only phos pholipid molecules in the outer half of the bilayer are available for the Table 1 Some widely used approaches to investigating the transverse distribution of membrane proteins Approach Accessibility to proteases Accessibility to lactoperoxidase-catalyzed iodination Accessibility to antibodies, lectins, and other macromolecules with specific binding sites Accessibility to nonpenetrating substrates and effectors of enzyme activity Accessibility to other non penetrating reagents
Selected references 16,23,39,52-56 5,57,58 58-60 40,42 5,23,24,39,52, 55,58,61-71
Involvement in vectorial processes, e.g. vectorial release of product, H+ translocation Reconstitution of membranes from isolated components
2, 70
39,45,46
/
ENZYME TOPOLOGY OF INTRACELLULAR MEMBRANES
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exchange catalyzed by these proteins (80, 8 1), and such exchange is a nondisruptive process that would not be expected to alter membrane struc ture. OTHER METHODS Other methods used in studying membrane structure, as well as the dynamics of this structure, include infrared spectroscopy, circular dichroism, optical rotatory dispersion, and other spectroscopic approaches (5, 39, 82), fluorescent probes (83), electron spin resonance spectroscopy (28), deuterium nuclear magnetic resonance (84), and low angle X-ray diffraction (39, 85).
MITOCHONDRIA
Morphology
The mitochondrion is generally depicted as having an outer membrane, an intermembrane space, an inner membrane, and a matrix (see Figure 2). The inner membrane is folded (these folds are commonly called cristae) so that the membrane surface area is greatly enlarged in. relation to that of the outer membrane. Several aspects of the topology of mitochondrial membranes have been covered in recent reviews (12, 42, 86-92). The Outer Membrane
The essential step in the separation of outer and inner mitochondrial mem branes is preferential disruption of the outer membrane by physical, chemi cal, or enzymatic treatment (93-96). These and complementary techniques have allowed determination of the localization of most mitochondrial en zymes. Relatively few of these enzymes-including acyl-CoA synthetase, amine oxidase, NADH-cytochrome hs reductase, cytochrome bs, glycero phosphate acyltransferase, kynurenine 3-monooxygenase, lysophosphati date acyltransferase, phospholipase A2 (97), and most recently a non-heme iron sulfur protein (98)-have been localized to the outer membrane. Table 2
Methods for investigating the transverse distribution of membrane lipids Method
Treatment with purified phospholipases
Selected references
22,2 3,72-74
Treatment with nonpenetrating reagents
18,19,21,24
Use of phospholipid exchange proteins
37 , 38
Isolation of the outer half of the bilayer
75,76
Selective removal of components of the outer leaflet by means of detergent
Use of nonactin-K+ complex as a probe
77 78
Cytoplasmic surface Goigi.
30
PC
30
Inner mitochondrial membrane
PC
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15
15 o .c 0VI o .c C.
ky'sosomes
Microsomes
30
'+ o
30
30
PC
30
15
-:::!2
o
15
30
30 Luminal surface
Figure 1 Phospholipid asymmetry in the transverse plane of the Golgi, inner mitochondrial, microsomal, and lysosomal membranes. From (79). The isolated organelles were treated with phospholipase A2 or sphingomyelinase in the presence of bovine serum albumin. Phospholipids hydrolyzed under these conditions were considered to be on the cytoplasmic half of the membrane bilayer (upward in the figure), whereas unhydrolyzed phospholipids were localized to the inner half (down ward in the figure). Abbreviations used are PC, phosphatidyIcholinej PE, phos phatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; Sph, sphingomyelin; CL, cardiolipin. This figure is reproduced with the kind permission of the authors.
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This paucity of enzymes has made the outer mitochondrial membrane a much less interesting object for study than the inner membrane. In addition, there are technical difficulties in studying the outer membrane: this struc ture contains only a small percentage of the total mitochondrial mass; 80-95% of the total membrane-bound mitochondrial protein and over 90% of the total mitochondrial lipid is associated with the inner membrane (99). Thus yields are small and even small amounts of contamination from the inner membrane or by other subcellular particles may lead to serious ar tifacts. Nor does one have available a preparation of vesicles deriving from the outer membrane whose inside-outside orientation has been shown to be the opposite of that in intact mitochondria. Finally, the use of low-molecular-weight reagents or molecules that do not normally penetrate membranes is not possible in studies of the trans verse topology of the outer membrane because of its unusual permeability in vitro. It is generally stated that this membrane is freely permeable to molecules with molecular weights of several thousands (86). This permea bility does not seem to reflect composition-the outer membrane contains twice as much phospholipid on a protein basis as the inner membrane and also contains a significant amount of cholesterol, which is known to lower the permeability of artificial membranes to lipid-insoluble substances (86). Instead, it has been argued that the high permeability of the outer mem brane is an artifact (100), due perhaps to distension of this structure during preparation of the mitochondria. If, however, the outer mitochondrial membrane proves to be naturally permeable to rather large substances, this may reflect a remarkable topology. The Inner Membrane GENERAL FEATURES The inner mitochondrial membrane is the in tracellular membrane whose enzyme topology has been most extensively
INNER MEMBRANE_--f-t""•• _
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r----
INTERMEMBRANE SPACE
MATRIX
SONICATION 1 /
SUBMITOCHONDRIAL PARTICLE
Figure 2
A
MEMBRANE PROJECTIONS
� �
schematic representation of the morphology of the mitochondrion.
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investigated. This is because it is the site of the respiratory chain, the particulate and highly organized nature of which was recognized long before its association with the inner mitochondrial membrane was demon strated. In addition, related energy-transducing processes such as ATP synthesis, energy-linked nicotinamide nucleotide transhydrogenation, and various carrier-facilitated ion translocations are also catalyzed by proteins located on this membrane. An exhaustive list of inner membrane enzymes can be found in a recent handbook (97); see also Wainio (99) for a compre hensive list of early references (1729 references!). The inner mitochondrial membrane is relatively rich in protein, as are energy-transducing membranes in general ( 101). It consists of approxi mately 75% protein and 25% lipid (102). Sixty to 70% of the protein is "intrinsic" or "integral" (42), which explains the early observation (103) that extraction of the bulk of the membrane lipid does not alter the trilami nar appearance of this membrane in electron micrographs. The lipids of this membrane are about 90% phospholipids; the main species are phos phatidylcholine, phosphatidylethanolamine, and cardiolipin in approxi mate molar ratios of 4:3:2. Cardiolipin is present chiefly, if not exclusively, in the inner membrane (86). Characteristic of the mitochondrial inner membrane is a very low perme ability, even to relatively small molecules. Lipid-insoluble nonelectrolytes with a molecular weight above 100-- 1 50 are virtually incapable of crossing this membrane by passive diffusion; in the case of charged molecules, even such simple ions as H+, K+, Na+, and Cl- cannot readily diffuse across the inner membrane (86). Submitochondrial particles derived from the inner membrane by sonica tion have an orientation opposite that of intact mitochondria, i.e. the outer surface of most of these particles corresponds to the inner (matrix) surface in intact mitochondria (see Figure 2) (104). This is a fortunate circumstance for those interested in studying the topology of the mitochondrial inner membrane: the outer surface of this membrane can be investigated using intact mitochondria (because of the high permeability of the outer mem brane in vitro; see above), while the inner surface is accessible in submito chondrial particles. GENERAL ASPECTS OF LATERAL TOPOLOGY The role of membrane structure in the function of the multienzyme systems of electron transport and oxidative phosphorylation has been the object of many investigations and hypotheses over the years. The central problem concerns how these enzymes interact with one another and why they need a membrane for this interaction. Are they present as fixed assemblies or do they diffuse laterally, independently of one another, interacting only when they collide? Such
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ENZYME TOPOLOGY OF INTRACELLULAR MEMBRANES
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questions, now so familiar in studies of biological membranes, first arose from studies of mitochondrial electron transport. Because of the rapid, sequential events involved in oxidative phosphory lation, many workers in the field are attracted to the hypothesis that the components involved are organized into complexes (see below) and that these are in tum organized into multicomplex assemblies ( 105-107). Con sistent with this hypothesis is the calculation that only about 40% of the surface of the inner mitochondrial membrane is covered by a phospholipid bilayer, with the remainder occupied by protein (101, 108). This figure seemed to be consistent with estimates of the surface area that is free from the particles seen in freeze-fracture profiles, and would not leave much room for membrane fluidity, especially if the phospholipid molecules imme diately surrounding membrane proteins are less mobile than the bulk mem brane lipid. However, these calculations were based on total membrane protein. Recent calculations based on integral membrane protein suggest that two thirds of the membrane surface is occupied by the phospholipid bilayer and the rest by protein (12). Another indication that diffusion of protein components in the lateral plane of the inner mitochondrial membrane may be important for protein protein interactions comes from experiments performed in Hackenbrock's laboratory (44, 109). These experiments were performed on rat liver mito chondria and inner membrane preparations and combined scanning calo rimetry with freeze-fracture electron microscopy (34). Slow freezing of these preparations caused intramembrane particles (presumably integral proteins) to aggregate, leaving large smooth patches (presumably lipid bilayer) on the membrane, whereas after rapid freezing the distribution of intramembrane particles was random. The changes caused by slow freezing were readily and rapidly reversible. These and complementary experiments led to the conclusion that the inner (and outer) mitochondrial membrane is a two-dimensional fluid allowing lateral diffusion of its integral proteins under physiological conditions. The half-lives of some of the protein-protein interactions involved in oxidative phosphorylation are rather rapid, e.g. 2 msec for the oxidation of cytochrome c by its oxidase ( 1 10). Hackenbrock (44) calculates that lateral diffusion in the inner mitochondrial membrane may be rapid enough to allow such short half-times without postulating the existence of multicomplex assemblies. However, it should not be forgotten that the ability of lateral diffusion to account for protein-protein interac tions in the inner mitochondrial membrane does not necessarily mean that this is the main mechanism by which such interactions occur. Finally, and perhaps most probably, both lateral diffusion and the existence of mul ticomplex assemblies may have important roles to play in electron transport and A TP synthesis.
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GENERAL ASPECTS OF TRANSVERSE TOPOLOGY The transverse topology of the inner membrane of the mitochondrion is interesting in two major respects. First, is its relationship to the metabolic processes going on in the soluble phases on both sides of this membrane. The matrix is the site of the citric acid cycle, fatty acid oxidation, and various ancillary reactions, whereas glycolysis and many biosynthetic processes are carried out in the cytoplasm. Various shuttles and translocators in the inner mitochondrial membrane control the flow of metabolites involved in the transfer of reduc ing equivalents, and the transport of ATP, ADP, and inorganic phosphate between the two compartments ( 1 1 1, 1 12). Secondly, for functional reasons certain components of both the electron transport and ATPase (ATP-synthesizing) systems are vectorially oriented across the inner mitochondrial membrane, in accordance with the princi ples of chemiosmotic coupling ( 1 1 3). There is now evidence that the three coupling sites of the respiratory chain ( 1 1 4- 1 1 6), the ATPase system ( 1 17), and the nicotinamide nucleotide transhydrogenase ( 1 1 8) are all capable of generating a proton gradient and/or a membrane potential (negative inside) across the membrane (see 9 1 and 92 for reviews). Thus, each of these systems must be able to span the membrane. There are presently two models concerning the mechanism by which a proton gradient is generated across the inner mitochondrial membrane in connection with electron transport and ATP hydrolysis-(a) a direct in volvement of protons in reactions taking place at the catalytic sites of the components of the electron transport chain and ATPase system, and (b) an indirect (conformational) interaction between catalytic and proton translocating regions (subunits) of these components. These two mecha nisms, which are discussed in some detail in another chapter of this volume (1 1 9), have important topological implications. The direct mechanism, proposed ( 1 1 3, 1 20) and strongly supported (92) by Mitchell, visualizes the electron transport system as being organized in "redox loops" consisting of hydrogen and electron carriers alternately span ning the membrane. Figure 3A illustrates the three proton-translocating redox loops of the respiratory chain currently suggested by advocates of the direct mechanism. According to the indirect mechanism, protons are trans located by amino acid side-chains, rather than by the prosthetic groups of the enzymes. Acid and base groups on these side-chains would undergo reversible pK changes during electron transfer through the catalytic sites of the enzymes, thereby acting as proton pumps. Thus, although the proton translocating regions of the enzymes must span the membrane, the catalytic sites as such need not be oriented vectorially (cf 9 1). A possible arrangement of the catalytic sites of the components of the respiratory chain, based on topological investigations, is shown schematically in Figure 3R. The evi-
213
ENZYME TOPOLOGY OF INTRACELLULAR MEMBRANES B
A M-side
C-side
M-side
C-side
I
(NADH++H+
2H+
Further
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ENZYME TOPOLOGY OF
+946
INTRACELLULAR MEMBRANES Joseph W. DePierre and Lars Ernster Department of Biochemistry, Arrhenius Laboratory, University of Stockholm, S-106 91 Stockholm, Sweden
CONTENTS PERSPECTIVES AND SUMMARY .................. ... ...................... ......... ................... ... INTRODUCTION ........... ......... . . . ........................................ ...... ........ .. ................
202 203
Contemporary Concepts of Membrane Structure ... ..... ....... . . .. ..... .. ......... Methodology for Studying the Topology of Membranes .. .. ................. . ..... Enzyme topology in the lateral plane ...... ........................ ............. ............ ........... Enzyme topology in the transverse plane . ............................... .................... ........ Lipid topology .... ............................. ...... ..................... .............. ............................ Other methods ................................................... ............................ .......................
204 205 205 206 206 207
MITOCHONDRIA ................................................................................................ ........
207
Morphology ............. . ............................................................................... .. .. ........ The Outer Membrane ................................. ................ ........ ................................. The Inner Membrane .............. ........... ...................... ................... .......................
207 207 209 209 210 212 214 214 215 217 218 220 221 222 226 227 229 230
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General features ...... ................... ......... ........................................... ..................... General aspects of lateral topology ...... .................. ............ .................................. General aspects of transverse topology .................... ............................................ The respiratory chain, ATPase, and related energy-transducing systems ...... ... General features................................................................................................ NADH-Q reductase (Camp/ex I) ... .. ... ........... ............ .................... ........... .... Succinate-Q reductase (Complex II)................................................................ Ubiquinone ... ..... ....... ..................... .................. ...... ............ .......... ..... ................. QHrcytochrome c reductase (Complex III) ................................................ ... Cytochrome c .................................................................. ..................... ............. Cytochrome c oxidase (Comp/ex IV) ................ .............................................. Transhydrogenase ..................... ... ......... ... ...................................... ...... ........ .... ATPase .............................................................................................................. Ion trans/ocators .. .. ................. ... ................ ... ........... ................ ... ........ ... ... ..... Other enzymes ......... .......... ..... ... ..................................... ......... .......... ...... ... ...... .... .
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CHLOROPLASTS ........................... ...............................................................................
General Aspects........................................ .............. ............ ...............................' " ... Photosystems I and II and Coupling Factor CF} ............................................ Localization 0/ Chlorophyll .................................................................................. Interactions between the Photosystems and Chlorophyll-Protein Complexes................................................................................................ Stacking 0/ Thylakoids..........................................................................................
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231 231 233 235 236 236
THE ENDOPLASMIC RETICULUM ........................................................................
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General Aspects........................................................................................................ Topology in the Lateral Plane.............................................................................. Topology in the Transverse Plane........................................................................ Binding of Membrane Proteins............................................................................ Complexes? .............. . . . .. .. ... . ... .... . . . ...... . . .. . .. ... . . . .. . . .. . . .. ... .. ... .. . . . . . .. . . . . . . Relationship of Proteins to Lipids ...................................................................... Functional Consequences of Topology ................................................................
237
THE G OLGI APPARATUS..........................................................................................
246
General Aspects........................................................................................................ Topology in the Lateral Plane.............................................................................. Topology in the Transverse Plane........................................................................
247
THE NUCLEAR ENVELOPE .. . . . . . ...... . . . . . ....................................................................
249
C ONCLUSION.. ........................................................... ........ . . .. . . . . . . . ................................
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PERSPECTIVES AND SUMMARY
The functional importance of the intracellular membranes of eukaryotic cells has long been apparent. The isolation and characterization of these structures have played a key role in the spectacular progress toward bridg ing the gap between cell and molecule that has taken place during the last 30 years. However, appreciation of the intimate relationship that exists between the function and topology of intracellular membranes is still grow ing. It is fascinating to see how, as the attention of more and more investiga tors is focused on this relationship, new techniques for studying membrane topology are developed, precise questions are formulated, and the answers begin to take shape. The basic structure of virtually all biological membranes is now thought to be the phospholipid bilayer. But how are proteins bound to this bilayer? How deep do they penetrate? Do they have special interactions with the phospholipid molecules immediately surrounding them? Are proteins dis tributed homogeneously (i.e. randomly) in the plane of the bilayer (the lateral plane) and across the membrane (the transverse plane)? If not, why not? Do proteins move in the lateral and transverse planes of the mem brane? If so, how rapidly? If not, why not? Many of these questions have been formulated as a result of studies on energy-transducing membranes, i.e. the inner mitochondrial membrane, the
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chloroplast thylakoid membrane, and the plasma membrane of prokaryotes. An important breakthrough in this field was the realization that the en zymes involved in electron transport and ATP synthesis are capable of giving rise to and interacting through electrochemical gradients across the membrane, gradients that constitute a functional link between these pro cesses and the process of active transport. While these discoveries give a solid physical framework to our understanding of cellular energy transduc tion, the underlying chemical mechanisms are still poorly understood. Con tinued progress in this field will be critically dependent on determining the reaction mechanisms of the catalysts involved and their topological rela tionship to one another in the membrane, as well as the dynamics of this topology. One of the most important results of studies on the membrane topology of the endoplasmic reticulum is a model for how certain integral proteins bind to their membranes. Cytochrome b5, NADH-cytochrome b5 reduc tase, and probably NADPH-cytochrome c reductase all have hydrophobic tails especially designed for anchoring them to the phospholipid bilayer. In addition, all enzymes whose distributions in the lateral and transverse planes of the endoplasmic reticulum have been carefully investigated have been found to be heterogeneously distributed. In most cases the structural and functional reasons for such distributions are not understood. Since the membrane of this organelle also contains two electron-transport chains (the cytochrome h5 system and the cytochrome P-450 system), evidence for the existence of macromolecular complexes has been much sought after; but to date, the evidence obtained cannot be unambiguously interpreted. Topological studies of the membrane of the Golgi apparatus and of the nuclear envelope have just begun, and the topologies of other organelles such as lysosomes and peroxisomes are still unknown. The future not only will hold further attempts to answer questions such as those formulated above but also will bring new questions about the details of the structure of the hydrophobic portion of the membrane, the dynamics of membrane topology, the interactions between different intracellular membrane sys tems, and the biogenesis of membranes. To answer them will require, as always, continued interest, ingenuity, and a little impatience. INTRODUCTION
Eukaryotic cells contain a variety of membranous organelles, at least some of which are probably the descendants of prokaryotic ancestors (1). The mitochondrion, the chloroplast, the endoplasmic reticulum, the Golgi ap paratus, the nucleus, the lysosome, and the peroxisome are examples of such
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organelles. Their membranes separate gene replication and transcription from gene translation, glycolysis from respiration and photosynthesis, in tracellular processes from the completion and transportation of products destined for secretion, etc. This compartmentation of cellular functions is of fundamental importance for the regulation of growth and differentiation and was presumably necessary for the evolution of multicellular organisms. As boundaries between different cellular compartments, the membranes of organelles are the sites where interactions between these compartments take place. A number of proteins, in particular transport (and "receptor"?) proteins, have evolved to control these interactions. In addition, intracellu lar membranes contain a variety of proteins with other functions as well, e.g. the electron-transport chains of the mitochondrion, chloroplast, and endoplasmic reticulum, and the enzymes of steroid and phospholipid me tabolism. In some cases the protein components of these metabolic path ways interact directly without mobile low-molecular-weight intermediates, and the membrane apparently maintains these components properly ori ented toward one another. In other cases the substrate of an enzyme may be part of the membrane itself or may "dissolve" in the bilayer; it is appar
ently efficient to have such an enzyme bound to the membrane. In still other instances, such as in energy-transducing membranes, enzymes may interact "chemiosmotically" by way of electrochemical gradients across the mem brane. By some as yet poorly understood process, biological membranes are assembled with a precise, asymmetric topology that is vital to the perfor mance of their cellular functions. This chapter is a survey of our current knowledge of the enzyme topology of intracellular membranes. To make it manageable, we have found it necessary to exclude the cell membrane [which has often been reviewed elsewhere (2-6)] and to exclude intracellular membranes of a rather special ized nature, such as synaptic vesicles, chromaffin granules, or the sarcoplas
mic reticulum. In addition, there is very little known about the enzyme topology of the membranes of a number of common organelles, such as lysosomes and peroxisomes. Here we discuss mitochondria, chloroplasts, the endoplasmic reticulum, the Golgi apparatus, and, very briefly, the nu clear envelope. We begin with a synopsis of current concepts of membrane structure and a survey of methods employed for the study of membrane topology. Contemporary Concepts of Membrane Structure
It is now generally accepted that most of the phospholipid in most biological membranes is arranged in a bilayer with the polar head groups at the two surfaces. A more controversial question is the extent to which this bilayer is interrupted by membrane proteins. These proteins are divided into two
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categories (7-13): peripheral (or extrinsic) proteins, which are thought to lie largely or completely on the aqueous side of the plane defined by the polar head groups of membrane phospholipids; and integral (or intrinsic) proteins, which are thought to pass through this plane and to make substan tial contact with the hydrocarbon region of the membrane. The bulk of the lipid in biological membranes seems to have no special structural relation ship to the membrane proteins (e.g. 9). However, results obtained with certain membrane proteins [for example, cytochrome c oxidase and rho dopsin (12)] suggest that they may have important structural interactions with the phospholipid molecules immediately surrounding them. It is now clear that most, if not all, peripheral and integral proteins are asymmetri cally distributed in the transverse plane of the membrane, and often in the lateral plane as well (3, 10, 1 4-16; see also below). Only recently has it become clear that different phospholipid species are also asymmetrically distributed in the transverse plane of the membrane (17-24). The structure of biological membranes is in a constant state of flux. Under physiological conditions most of the lipids diffuse rather freely in the plane of the membrane (13, 25-30) and their hydrocarbon chains are disor dered (10, 26, 27, 30-33). A number of membrane proteins also seem to diffuse rather rapidly in the lateral plane of the bilayer (34, 35), a finding that has given rise to the fluid mosaic model of membrane structure (9, II, 12, 36). One the other hand, transverse rotation of phospholipids ("flip flop") and of membrane proteins is apparently a very slow process (34, 35, 37, 38). Methodology for Studying the Topology of Membranes ENZYME TOPOLOGY IN THE LATERAL PLANE At present, the most widely used techniques for investigation of enzyme topology in the lateral plane of membranes are subfractionation (including disruption of the mem brane into smaller pieces) (6, 39-41), normal (39, 42) and freeze-fracture (10, 34, 43, 44) electron microscopy, and reconstitution of membranes from isolated components (39, 45, 46). Other techniques have been developed e.g. cross-linking of membrane proteins to one another (47-49) and photo lysis-induced cross-linking of the fatty acid moiety of phospholipids to pro teins in their immediate vicinity (50, 5 1 )-and should find broader application. The first of these two new methods should allow "nearest neighbor analysis" of membrane proteins, while the latter should allow investigation of possible specific associations between membrane proteins and the phospholipid molecules immediately surrounding them, as well as determination of which parts of a protein extend down into the hydrophobic region of the membrane.
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ENZYME TOPOLOGY IN THE TRANSVERSE PLANE Some of the most widely used approaches for determining the transverse distribution of mem brane proteins are listed in Table 1. As with all experimental techniques, these approaches involve hazards of which the investigator must be con stantly aware (2, 40). For instance, many of these methods utilize sub stances (proteases, lactoperoxidase, antibodies, small charged molecules) to which membranes are supposed to be impermeable. This impermeability should never be assumed, but should always be demonstrated. On the other hand, many of these approaches are reliable only when a positive result is obtained. For example, if a protease or antibody or nonpenetrating reagent fails to inhibit an enzyme present on the membrane of a closed vesicle, it must be shown that these agents do inhibit the enzyme when the vesicle is opened, before any conclusions can be drawn. LIPID TOPOLOGY The topology of membrane lipids is almost certainly related-biogenically and/or functionally-to the topology of membrane proteins. Table 2 lists some of the methods that have been used to study the transverse distribution of membrane lipids in biological membranes. Of these approaches, treatment with purified phospholipases has been the most widely used and is the only method whose application to intracellular membranes has been reported (79). This study revealed that phospholipid species are asymmetrically distributed in the transverse plane of the mem brane of microsomes, the inner mitochondrial membrane, the Golgi mem brane, and the lysosomal membrane (Figure 1). In the future, phospholipid exchange proteins will surely be more widely used in studies of phospholipid distribution in membranes. Only phos pholipid molecules in the outer half of the bilayer are available for the Table 1 Some widely used approaches to investigating the transverse distribution of membrane proteins Approach Accessibility to proteases Accessibility to lactoperoxidase-catalyzed iodination Accessibility to antibodies, lectins, and other macromolecules with specific binding sites Accessibility to nonpenetrating substrates and effectors of enzyme activity Accessibility to other non penetrating reagents
Selected references 16,23,39,52-56 5,57,58 58-60 40,42 5,23,24,39,52, 55,58,61-71
Involvement in vectorial processes, e.g. vectorial release of product, H+ translocation Reconstitution of membranes from isolated components
2, 70
39,45,46
/
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exchange catalyzed by these proteins (80, 8 1), and such exchange is a nondisruptive process that would not be expected to alter membrane struc ture. OTHER METHODS Other methods used in studying membrane structure, as well as the dynamics of this structure, include infrared spectroscopy, circular dichroism, optical rotatory dispersion, and other spectroscopic approaches (5, 39, 82), fluorescent probes (83), electron spin resonance spectroscopy (28), deuterium nuclear magnetic resonance (84), and low angle X-ray diffraction (39, 85).
MITOCHONDRIA
Morphology
The mitochondrion is generally depicted as having an outer membrane, an intermembrane space, an inner membrane, and a matrix (see Figure 2). The inner membrane is folded (these folds are commonly called cristae) so that the membrane surface area is greatly enlarged in. relation to that of the outer membrane. Several aspects of the topology of mitochondrial membranes have been covered in recent reviews (12, 42, 86-92). The Outer Membrane
The essential step in the separation of outer and inner mitochondrial mem branes is preferential disruption of the outer membrane by physical, chemi cal, or enzymatic treatment (93-96). These and complementary techniques have allowed determination of the localization of most mitochondrial en zymes. Relatively few of these enzymes-including acyl-CoA synthetase, amine oxidase, NADH-cytochrome hs reductase, cytochrome bs, glycero phosphate acyltransferase, kynurenine 3-monooxygenase, lysophosphati date acyltransferase, phospholipase A2 (97), and most recently a non-heme iron sulfur protein (98)-have been localized to the outer membrane. Table 2
Methods for investigating the transverse distribution of membrane lipids Method
Treatment with purified phospholipases
Selected references
22,2 3,72-74
Treatment with nonpenetrating reagents
18,19,21,24
Use of phospholipid exchange proteins
37 , 38
Isolation of the outer half of the bilayer
75,76
Selective removal of components of the outer leaflet by means of detergent
Use of nonactin-K+ complex as a probe
77 78
Cytoplasmic surface Goigi.
30
PC
30
Inner mitochondrial membrane
PC
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15
15 o .c 0VI o .c C.
ky'sosomes
Microsomes
30
'+ o
30
30
PC
30
15
-:::!2
o
15
30
30 Luminal surface
Figure 1 Phospholipid asymmetry in the transverse plane of the Golgi, inner mitochondrial, microsomal, and lysosomal membranes. From (79). The isolated organelles were treated with phospholipase A2 or sphingomyelinase in the presence of bovine serum albumin. Phospholipids hydrolyzed under these conditions were considered to be on the cytoplasmic half of the membrane bilayer (upward in the figure), whereas unhydrolyzed phospholipids were localized to the inner half (down ward in the figure). Abbreviations used are PC, phosphatidyIcholinej PE, phos phatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; Sph, sphingomyelin; CL, cardiolipin. This figure is reproduced with the kind permission of the authors.
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This paucity of enzymes has made the outer mitochondrial membrane a much less interesting object for study than the inner membrane. In addition, there are technical difficulties in studying the outer membrane: this struc ture contains only a small percentage of the total mitochondrial mass; 80-95% of the total membrane-bound mitochondrial protein and over 90% of the total mitochondrial lipid is associated with the inner membrane (99). Thus yields are small and even small amounts of contamination from the inner membrane or by other subcellular particles may lead to serious ar tifacts. Nor does one have available a preparation of vesicles deriving from the outer membrane whose inside-outside orientation has been shown to be the opposite of that in intact mitochondria. Finally, the use of low-molecular-weight reagents or molecules that do not normally penetrate membranes is not possible in studies of the trans verse topology of the outer membrane because of its unusual permeability in vitro. It is generally stated that this membrane is freely permeable to molecules with molecular weights of several thousands (86). This permea bility does not seem to reflect composition-the outer membrane contains twice as much phospholipid on a protein basis as the inner membrane and also contains a significant amount of cholesterol, which is known to lower the permeability of artificial membranes to lipid-insoluble substances (86). Instead, it has been argued that the high permeability of the outer mem brane is an artifact (100), due perhaps to distension of this structure during preparation of the mitochondria. If, however, the outer mitochondrial membrane proves to be naturally permeable to rather large substances, this may reflect a remarkable topology. The Inner Membrane GENERAL FEATURES The inner mitochondrial membrane is the in tracellular membrane whose enzyme topology has been most extensively
INNER MEMBRANE_--f-t""•• _
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schematic representation of the morphology of the mitochondrion.
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investigated. This is because it is the site of the respiratory chain, the particulate and highly organized nature of which was recognized long before its association with the inner mitochondrial membrane was demon strated. In addition, related energy-transducing processes such as ATP synthesis, energy-linked nicotinamide nucleotide transhydrogenation, and various carrier-facilitated ion translocations are also catalyzed by proteins located on this membrane. An exhaustive list of inner membrane enzymes can be found in a recent handbook (97); see also Wainio (99) for a compre hensive list of early references (1729 references!). The inner mitochondrial membrane is relatively rich in protein, as are energy-transducing membranes in general ( 101). It consists of approxi mately 75% protein and 25% lipid (102). Sixty to 70% of the protein is "intrinsic" or "integral" (42), which explains the early observation (103) that extraction of the bulk of the membrane lipid does not alter the trilami nar appearance of this membrane in electron micrographs. The lipids of this membrane are about 90% phospholipids; the main species are phos phatidylcholine, phosphatidylethanolamine, and cardiolipin in approxi mate molar ratios of 4:3:2. Cardiolipin is present chiefly, if not exclusively, in the inner membrane (86). Characteristic of the mitochondrial inner membrane is a very low perme ability, even to relatively small molecules. Lipid-insoluble nonelectrolytes with a molecular weight above 100-- 1 50 are virtually incapable of crossing this membrane by passive diffusion; in the case of charged molecules, even such simple ions as H+, K+, Na+, and Cl- cannot readily diffuse across the inner membrane (86). Submitochondrial particles derived from the inner membrane by sonica tion have an orientation opposite that of intact mitochondria, i.e. the outer surface of most of these particles corresponds to the inner (matrix) surface in intact mitochondria (see Figure 2) (104). This is a fortunate circumstance for those interested in studying the topology of the mitochondrial inner membrane: the outer surface of this membrane can be investigated using intact mitochondria (because of the high permeability of the outer mem brane in vitro; see above), while the inner surface is accessible in submito chondrial particles. GENERAL ASPECTS OF LATERAL TOPOLOGY The role of membrane structure in the function of the multienzyme systems of electron transport and oxidative phosphorylation has been the object of many investigations and hypotheses over the years. The central problem concerns how these enzymes interact with one another and why they need a membrane for this interaction. Are they present as fixed assemblies or do they diffuse laterally, independently of one another, interacting only when they collide? Such
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questions, now so familiar in studies of biological membranes, first arose from studies of mitochondrial electron transport. Because of the rapid, sequential events involved in oxidative phosphory lation, many workers in the field are attracted to the hypothesis that the components involved are organized into complexes (see below) and that these are in tum organized into multicomplex assemblies ( 105-107). Con sistent with this hypothesis is the calculation that only about 40% of the surface of the inner mitochondrial membrane is covered by a phospholipid bilayer, with the remainder occupied by protein (101, 108). This figure seemed to be consistent with estimates of the surface area that is free from the particles seen in freeze-fracture profiles, and would not leave much room for membrane fluidity, especially if the phospholipid molecules imme diately surrounding membrane proteins are less mobile than the bulk mem brane lipid. However, these calculations were based on total membrane protein. Recent calculations based on integral membrane protein suggest that two thirds of the membrane surface is occupied by the phospholipid bilayer and the rest by protein (12). Another indication that diffusion of protein components in the lateral plane of the inner mitochondrial membrane may be important for protein protein interactions comes from experiments performed in Hackenbrock's laboratory (44, 109). These experiments were performed on rat liver mito chondria and inner membrane preparations and combined scanning calo rimetry with freeze-fracture electron microscopy (34). Slow freezing of these preparations caused intramembrane particles (presumably integral proteins) to aggregate, leaving large smooth patches (presumably lipid bilayer) on the membrane, whereas after rapid freezing the distribution of intramembrane particles was random. The changes caused by slow freezing were readily and rapidly reversible. These and complementary experiments led to the conclusion that the inner (and outer) mitochondrial membrane is a two-dimensional fluid allowing lateral diffusion of its integral proteins under physiological conditions. The half-lives of some of the protein-protein interactions involved in oxidative phosphorylation are rather rapid, e.g. 2 msec for the oxidation of cytochrome c by its oxidase ( 1 10). Hackenbrock (44) calculates that lateral diffusion in the inner mitochondrial membrane may be rapid enough to allow such short half-times without postulating the existence of multicomplex assemblies. However, it should not be forgotten that the ability of lateral diffusion to account for protein-protein interac tions in the inner mitochondrial membrane does not necessarily mean that this is the main mechanism by which such interactions occur. Finally, and perhaps most probably, both lateral diffusion and the existence of mul ticomplex assemblies may have important roles to play in electron transport and A TP synthesis.
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GENERAL ASPECTS OF TRANSVERSE TOPOLOGY The transverse topology of the inner membrane of the mitochondrion is interesting in two major respects. First, is its relationship to the metabolic processes going on in the soluble phases on both sides of this membrane. The matrix is the site of the citric acid cycle, fatty acid oxidation, and various ancillary reactions, whereas glycolysis and many biosynthetic processes are carried out in the cytoplasm. Various shuttles and translocators in the inner mitochondrial membrane control the flow of metabolites involved in the transfer of reduc ing equivalents, and the transport of ATP, ADP, and inorganic phosphate between the two compartments ( 1 1 1, 1 12). Secondly, for functional reasons certain components of both the electron transport and ATPase (ATP-synthesizing) systems are vectorially oriented across the inner mitochondrial membrane, in accordance with the princi ples of chemiosmotic coupling ( 1 1 3). There is now evidence that the three coupling sites of the respiratory chain ( 1 1 4- 1 1 6), the ATPase system ( 1 17), and the nicotinamide nucleotide transhydrogenase ( 1 1 8) are all capable of generating a proton gradient and/or a membrane potential (negative inside) across the membrane (see 9 1 and 92 for reviews). Thus, each of these systems must be able to span the membrane. There are presently two models concerning the mechanism by which a proton gradient is generated across the inner mitochondrial membrane in connection with electron transport and ATP hydrolysis-(a) a direct in volvement of protons in reactions taking place at the catalytic sites of the components of the electron transport chain and ATPase system, and (b) an indirect (conformational) interaction between catalytic and proton translocating regions (subunits) of these components. These two mecha nisms, which are discussed in some detail in another chapter of this volume (1 1 9), have important topological implications. The direct mechanism, proposed ( 1 1 3, 1 20) and strongly supported (92) by Mitchell, visualizes the electron transport system as being organized in "redox loops" consisting of hydrogen and electron carriers alternately span ning the membrane. Figure 3A illustrates the three proton-translocating redox loops of the respiratory chain currently suggested by advocates of the direct mechanism. According to the indirect mechanism, protons are trans located by amino acid side-chains, rather than by the prosthetic groups of the enzymes. Acid and base groups on these side-chains would undergo reversible pK changes during electron transfer through the catalytic sites of the enzymes, thereby acting as proton pumps. Thus, although the proton translocating regions of the enzymes must span the membrane, the catalytic sites as such need not be oriented vectorially (cf 9 1). A possible arrangement of the catalytic sites of the components of the respiratory chain, based on topological investigations, is shown schematically in Figure 3R. The evi-
213
ENZYME TOPOLOGY OF INTRACELLULAR MEMBRANES B
A M-side
C-side
M-side
C-side
I
(NADH++H+
2H+
