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The structure of biological membranes in relation to the principle of energy coupling
J. theor. Biol(1976)
62, 271-285
The Structure of Biological Membranes in Relation to the Principle of Ehergy Coupling D. E. GREEN Institute for Emzyme Research, University of Wisconsin, Madison, Wisconsin 53706, U.S.A.
The thesis that the structure of biological membranes translates the principle of energy coupling has been systematically developed. The protein ribbon continuum has been identified as the structural feature of membranes which is relevant to this translation. The body of evidence bearing on the widespread occurrence of ribbon structure in membranes has been reviewed. Fusion of membranes, the crystalline state of membranes, as well as the extensive cross-linking of membrane proteins are phenomena predictable from ribbon structure. The possible ways in which ribbon structure in membranes can effectively translate energy coupling, the control of energy coupling, and the immune response have been considered. 1. Introduction
The case for postulating a universal principle of energy that underlies all bioenergetic phenomena has been developed by Green (Green, 1974a,b; Blondin & Green, 1975; Green & Reible, 1974; Green & Reible, 1975; Green, Blondin, Kessler & Southard, 1975) and by Kemeny (Kemeny, 1974u,b; Kemeny & Singer, 1975). In the present article, we shall be considering the implications of this universality of the energy principle for the structure of biological membranes. Since membranes are the instruments of a wide variety of coupled processes (active transport of cations and metabolites, oxidative and photosynthetic phosphorylation, coupled electron flow and hydrolysis of ATP), we may presume that the structure of membranes is appropriate for translating the energy principle that underlies these coupled processes. In fact, we may go further and assert that the struoture of membranes which carry out coupled functions has to embody features which are essential for this translation. Since membrane function is invariably subject to control, the structure of membranes must satisfy two requirements-translation of the function, as well as translation of the control of 271
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this function. This means that all membranes which carry out some form of energy coupling must have structural features in common that are requisite for translating the control and functional parameters of energy coupling. The often repeated cliche that no two membranes are alike should be recognized as a highly dubious assertion, since it denies the limitations on the variability of structure imposed by the requirements for function and control. One must distinguish between variability in respect to details and variability in respect to fundamentals. Certainly there will be variability at the level of chemical and morphological detail, but it is also obvious that variability in respect to the structural features requisite for translating the energy principle and the control of energy coupling will necessarily be severely circumscribed. 2. The Ribbon Structure of the Mitochondrion The structural feature of membranes which we consider most relevant to energy coupling was discovered by Fernandez-Moran, Oda, Blair & Green (1964) in 1961 in their studies of negatively stained mitochondria. They observed paired and closely apposed strips of inner membrane with arrays of headpiece-stalks projecting in one direction on one side of the fused strips and projecting in the opposite direction on the other side. The obvious conclusion to be drawn from these electron micrographs was that the inner membrane had a continuous ribbon structure with headpiece-stalks projecting from the ribbon in precise periodicity. This periodicity showed up sharply when two strips of membrane were fused together-bottom to bottom. Such a simple and obvious interpretation was not drawn, however, because it would have been completely out of line with the then current dogma as to the structure of membranes. Instead, the arrays were considered to be apparent arrays in the same sense that individual tombstones in a cemetery appear as arrays of tombstones when viewed from a sufficient distance. Subsequently, such orderly arrays of projecting headpiece-stalks were found in a wide variety of membranes-sarcoplasmic reticulum (Ikimoto, Streeter, Nakamura & Gergely, 1968; Inesi & Asai, 1968), microsomal membranes (MacLennan, Ostwald & Stewart, 1967), microvilli (Oda & Seki, 1966), chloroplast (Oleszko & Moudrianakis, 1974), plasma membranes (Benedetti & Emmelot, 1965) and viral membranes (Dales & Mosbach, 1968)-and with monotonous regularity, these arrays were rationalized as apparent arrays. F. Crane (Crane, Stiles, Prezbindowski, Rujicka & Sun, 1968) was one of the few to insist on the reality of the continuous array. He produced electron microscopic evidence in negatively stained preparations of mitochondria and submitochondrial particles that
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detergent such as Triton X-100 induced the formation of large numbers of iilamentous structures with arrays of projecting headpiece-stalks. In the study of electron transport particles exposed to the fragmenting action of lysolecithin, Sadler, Hunter & Haworth (1974) found in negatively stained preparations of such treated particles numerous examples of open-ended strips of ribbon structure with arrays of headpiece-stalk projections (see Fig. 1 for a drawing of such structures). In this case, any interpretation in
FIG. 1. A drawing representativeof the ribbon strips observed by Sadler et-al.(1974) in electron micrographs of negatively stained lysolecithin-treated electron transport particles. The cross-section of the ribbon is intended to emphasize the non-membranous character of the ribbon. terms of stacking and apparent arrays could be definitively excluded, since the structures visualized were no longer closed membranes, but fragments of membranes. It was then obvious that a continuous ribbon structure was formed by fragmentation of the inner mitochondrial membrane and that this ribbon structure could not be lipoidal in nature, since lysolecithin effectively eliminated bilayer structure. The continuous, but segmented, structure to which the arrays of headpiece-stalks were attached had to be predominantly protein in nature. The proof of this has been established by direct isolation of the segmented units (Sadler et al., 1974). These continuous structures we have designated as ribbons. The next order of business was to find conditions and reagents which would induce ribbon formation in an across-the-board fashion. The experimental aim was to “ribbonize” the entire inner membrane and to stabilize the membrane in this ribbon&d state. Our first efforts (Haworth and Green, unpublished studies) were directed to a search for conditions which favored this ribbonization of the mitochondrial inner membrane. We found that ribbonization was favored with frozen mitochondria that were thawed under hypotonic conditions at pH 8. To a high degree, such mitochondria
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FIG. 2. A drawing representative of the continuum observed in electron micrographs of negatively stained beef heart mitochondria after freezing, thawing and exposure to alkaline pH (c. 8-O). The drawing of the cross-section of the continuum emphasizes that the structure is a tubular membrane and that there are two ribbons per tubule, separated one from another by a zone of bilayer phospholipid. The electron micrographs from which the drawings were made were prepared by R. A. Haworth.
showed extended ribbons with paired arrays of headpiece-stalks. These ribbons were not membrane fragments, as in the case of lysolecithin-treated particles. They were, in fact, tubular membranes with paired ribbon structure as depicted diagrammatically in Fig. 2. Bilayer phospholipid separated the two ribbons in the tubular membrane. The occurrence of ribbon structure within the intact membrane suggests a possible explanation for the hitherto bizarre observation of cristae showing regular geometrical shape. When the number is two, the tubule is spherical; when the number is three, the tubule is triangular (Blinzenger, Rewcastle & Hagar, 1965); and when the number is four, the tubule has a square crosssectional appearance (Fain-Maurel, 1968). In mitochondria from different sources with tubular cristae, one can readily see these variations in the geometry of the tubular cristae. Once it was recognized that tubularization of the inner membrane is equivalent to ribbonization, some earlier unpublished studies in our laboratory by J. Smoly and T. Wakabayashi came into clearer perspective. These investigators found that silicotungstate induced quantitative tubularization of the inner mitochondrial membrane. The headpiece-stalks in the spherical tubules induced by silicotungstate projected into the interior instead of exteriorily, as in the case of the lysolecithin-induced ribbons. Furthermore, the diameter of the silicotungstate-induced tubules was considerably larger (3-4 times) than the tubules induced by exposure of previously frozen mitochondria to hypotonic alkaline conditions-a token that the number of ribbons per tubule would be at least six.
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The long and short of these experimental studies of Haworth and Green is that they established the reality of ribbon structure in mitochondria and opened the door to phenomena derivative from ribbon structure-phenomena such as tubularization. Apparently, there is a dynamic equilibrium between the planar state of the crista and the tubular&d state in which one crista gives rise to a number of tubules all interconnected. There apparently is also a dynamic equilibrium between extended ribbons and short strips of ribbon. We deduce this from the fact that the ribbons of enormous length seen in tubular cristae of hypotonic mitochondria must have arisen by rearrangement and end-to-end association of shorter structures in the planar membrane from which they are derived. 3. Structural Organization
of the Ribbon Continuum
We may define the ribbon structure of the rnitochondrial inner membrane in the following terms. It is a protein continuum which, like bilayer phospholipid, constitutes a section of the membrane. The inner membrane is thus a composite of a protein domain (the ribbon) and a phospholipid domain (the bilayer zone). The ribbons are separated one from another by a zone of bilayer phospholipid. The cross-sectional diameter of the ribbon, i.e. from one side of the membrane to the other, is about 60-75 A; the comparable dimension for the bilayer is about 50 A. Thus, the protein domain of the membrane is always slightly thicker than the phospholipid domain. The second cross-sectional dimension of the ribbon is about 120 A-wide enough to accommodate a foursome of elongated proteins, each with a diameter of about 30 A. The proteins may extend the width of the membrane (through membrane proteins) or half-way across the membrane (like the phospholipids in the bilayer). The bonds which hold together the proteins in the ribbon structure are predominantly hydrophobic in nature (Komai, Hunter & Takahashi, 1973). Lysolecithin can fragment the segmented ribbon into smaller unitscommonly with two headpiece-stalk projections (Komai et al., 1973). One such unit has a length of about 300 A and in cross-section, the unit would have the geometry of a rectangle 75 x 60 A. The ribbon is apparently formed by the hydrophobic association of linear sets of units, each with two headpiece-stalk projections. 4. Functional Parameters of the Ribbon Continuum The entire energy coupling apparatus of the mitochondrion is localized in the ribbon structure of the inner membrane. This would include the
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systems for active transport of cations, for oxidative phosphorylation, for electron transfer and ATP hydrolysis, and for energized transhydrogenation. We may reasonably assume that mitochondrial coupling functionality is segmented and that each unit of the ribbon with two headpiece-stalk projections has the capacity for all the above-listed coupled functions. Sadler et al. (1974) have shown that lysolecithin strips the electron transfer chain cleanly away from the ribbon structure to which the headpiece-stalk projections are attached, whereas the ribbons in tubularized membranes have their full complement of the electron transfer complexes. These observations suggest that the protein ribbon in the intact inner membrane is a composite of two ribbons-one containing the complexes of the electron transfer chain (the electron transfer chain ribbon) and the other containing the segmented units with headpiece-stalk projections (the tripartite ribbon). These two ribbons would be tightly apposed laterally forming, in effect, a fusion ribbon. The individual ribbons would have a diameter of about 60 A-wide enough to accommodate two elongated proteins, each with a diameter of about 30 A. 5. Translation of Energy Coupling by the Ribbon Continuum Now we may pose the critical question whether ribbon structure has special advantages in respect to the facilitation of energy coupling. We have postulated that the essence of energy coupling is paired charge separation, paired charge movement and paired charge elimination (Blondin & Green, 1975). This requires the parallel and vectorial flow of two charged species, each in a separate reaction center, across the membrane (Blondin 62 Green, 1975). The close apposition of the electron transfer chain ribbon and the tripartite ribbon provides the ideal combination for such paired charge flow (Fig. 3). Since the two moving species must be as close as possible for maximization of coupling, the suitability of the ribbon modality for achieving such close proximity is obvious. This proximity applies not only to the interaction between a moving ion in the electron transfer chain ribbon and a moving ion in the tripartite ribbon, but also to the interaction between paired moving ions, both of which are in the tripartite ribbon. There are two features of ribbon structure that are particularly relevant to the necessities for energy coupling. The first is the straight line geometry of the ribbon in the long dimension and the rectangular geometry in crosssection. For tight apposition of coupled complexes, for minimization of the distance between complexes, and for precise vectorial movements of the two coupled charge species, the geometries inherent in ribbon structure are ideally suited for the requirements of energy coupling. The second feature
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FIG. 3. A drawing illustrating how electron transfer can be.coupled to synthesis of ATP in the ribbon continuum. The flow of the electron in the electron transfer ribbon is vectorially aligned to the flow of the positively charged ionophoric species in the tripartite ribbon. The two ribbons are tightly apposed and in exact register.
has to do with the problem of alignment of complexes in the electron transfer chain ribbon with complexes in the tripartite ribbon. Since the electron transfer chain ribbon has to move with respect to the tripartite ribbon as each electron transfer complex is successively brought into position for coupling with the counterpart complex in the tripartite unit, this would involve the sliding of one ribbon with respect to the other. For such a sliding maneuver, the linear and rectangular geometry of the ribbon would be a great advantage. The advantages of ribbon structure for energy coupling that we have listed should be independent of the length of the ribbon. What, then, would be the advantage of extended ribbon structure for energy coupling? Experiment has shown that the efficiency of energy coupling is greatly reduced when ribbons are fragmented to successively smaller units (Sadler et al., 1974). The 32Pi-ATP exchange activity is an order of magnitude lower on a specific activity basis for the same unit in isolation as compared to the unit which is part of extended ribbon structure of the mitochondrion (Sadler et al., 1974). This observation would suggest that the length of the ribbon may be an important factor in the efficiency of coupling. Chance & Yoshioka (1966) have shown that mitochondria can undergo oscillations in a remarkably regular pattern-oscillations involving the inflow and outflow of ions into the mitochondrion. This regularity bespeaks the synchronizing of metabolic activities within individual mitochondria. It is as if, at a given time, the same complex in each electron transfer chain in a mitochondrion is engaged in coupling. This type of synchronization requires a built-in structural feature and we are proposing that ribbon structure provides this very
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feature. Why synchronization is a desirable property is still an unanswered question, but that it is present in mitochondria is undisputable. During energy coupling, there is interplay between the matrix proteins, the coupling systems of the inner membrane, and the mechanisms for translocation of metabolites. This type of interplay which is part and parcel of synchronization would certainly call for the type of cooperative behavior that only an extended structure like a ribbon should show. 6. Control of Energy Coupling by the Ribbon Continuum The mitochondrion has a control mechanism which regulates the pattern of coupling capability (unpublished studies of Hunter, Haworth and Southard). Mg ‘+ favors, or induces, the state appropriate for oxidative phosphorylation and high-affinity transport of Ca’+ and Mg2+ ; Ca2+ favors, or induces, the state in which the mitochondrion lacks the ability to carry out oxidative phosphorylation, but is capable of low-a6nity active transport of Ca’+, Mg2+ and K+ (Southard & Green, 1974). The Mg2+favored state corresponds to the aggregated configuration, and the Ca2+-favored state to the orthodox configuration of the mitochondrion. In the aggregated configuration, the headpiece-stalk projection appears to be retracted into the membrane; in the orthodox configuration, the headpiece-stalk is known to be extended from the membrane (Hatase, Wakabayashi, Hayashi & Green, 1972). The control mechanism of the mitochondrion very likely controls the contraction and extension of the stalk which thereby controls the movement in and out of the membrane of the headpiece (F,). Concomitant with the contraction and extension of the stalk, there is not only the movement of the headpiece (F,) in and out of the membrane, but also a change in the pattern of energy coupling and a change in the permeability of the membrane to neutral solutes such as sugars. In consequence of these changes which accompany the contraction and extension of the stalk, the cristae undergo a transition from one configurational state to another. The cristae expand and round up when the headpiece is inserted into the membrane (aggregated configuration); and contract and straighten out when the headpiece is ejected from the membrane (orthodox configuration). This type of control mechanism involves a change in state of all the cristae in the mitochondrion. Such a blanket change in the state of the mitochondrial cristae can be most effectively translated by extended ribbon structure. This control mechanism cannot be general for all membranes with projecting headpiece-stalks, since it is only in the mitochondrion, the chloroplast, and bacterial membranes that the ATPase and ATP synthetase are associated
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with the headpiece (ATPase and ATP synthetase are part of the headpiecestalk-basepiece ensemble). In other membranes (the membranes with microheadpiece-stalks), the ATPase is permanently intrinsic to the membrane and is never extruded (MacLennan et al., 1974). The headpiece of the viral membrane has been identsed as a neuramidase (Laver, 1973), the headpiece of the microvilli as a carbohydrate-splitting enzyme (Oda & Seki, 1966). Does the control mechanism in such membranes involve the contraction into, and extension from the membrane of the headpiece? If analogy is to be our guide, it would be predictable that the control mechanism in membranes with projecting microheadpieces would involve the same in-and-out movement of the head pieces and this maneuver would lead to two alternative states of the membrane. In one state, the membrane would have the enzymic capability intrinsic to the headpiece; in the other state, this capability would be absent. The appearance and disappearance of this capability in the membrane would be blanket for the entire organelle. Extended ribbon structure would insure that control would be an across-the-board, rather than a local phenomenon. The proof that headpiece-stalks can contract into the membrane is provided by the evidence of fusion. Two membranes with projecting headpiece-stalks can be induced to undergo fusion (unpublished studies of Wakabayashi and Green). After fusion, the space between the two membranes disappears. If the headpiece-stalk projections were not able to contract into the membrane, fusion of the two membranes would be impossible. It should be recalled that the headpiece-stalks project at least 150 A from the membrane (Fernandez-Moran et al., 1964). Without retraction of the headpieces, the two membranes would never get closer than 300 A apart. The in-and-out movement of the headpieces probably involves the contraction and extension of the stalk. This would mean that the stalk is a contractile protein and that the control maneuver regulates the ion-dependent reversible contraction and relaxation of this protein. Some rearrangement of the proteins in the headpiece would be required for this in-and-out maneuver of the membrane. In the membrane, the headpiece would have to expose a hydrophobic surface. Out of the membrane, the headpiece would have to expose a hydrophilic surface. This change in polarity would necessarily entail some rotation of the proteins in the headpiece. Shape changes in the ribbon continuum go parallel with the changes in configurational state induced by the control mechanism. A variety of membrane phenomena require or depend upon such shape changes. The change in shape of lymphocytes from the stationary to the mobile form is one dramatic example of an energy-requiring control mechanism (Unanue, 1974). The formation of pseudopodia by motile cells is yet another.
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7. How General is Ribbon Structure in Membranes? A crucial question is whether ribbon structure is an intrinsic feature of all membranes, or only of membranes with the capability for active transport or some other form of energy coupling. We are unaware of any biological membrane that shows no evidence of the capacity of energy coupling. The myelin membrane could be considered one such case, but it must be remembered that the myelin membrane is derived from the plasma membrane of the Schwann cell (Giren, 1954) and all plasma membranes unquestionably have the capacity for energy coupling. It is inconceivable that ribbon structure would be intrinsic to the myelin membrane in the early stages, but not in the later stages of differentiation. The relatively low concentration of protein in the myelin membrane (20 % versus 50% or more in other membranes) could be interpreted as a token that the structure of the myelin membrane is atypical (O’Brien 8z Sampson, 1965). The mass ratio of protein ribbon : bilayer phospholipid need not necessarily be 1 : 1 as in the mitochondrion. The zone of bilayer phospholipid in the myelin membrane separating one ribbon from another could be far wider than in other membranes. Hence, a low protein : phospholipid ratio may have no bearing on the presence or absence of ribbon structure. Ribbon structure is compatible with either a low or a high ratio. 8. The Crystalline State of Membranes in Relation to Ribbon Structure
A high proportion of biological membranes have been shown to exist in a checkerboard crystalline state (Green, 1972). This checkerboard crystallinity reflects the precise intercalation of protein and phospholipid domains as Vanderkooi & Copaldi (1972) have shown in two studies of crystalline cytochrome exidase (Maniloff, Vanderkooi, Hayashi & Capaldi, 1973; Hayashi et al., 1972). If, as we suspect, the crystalline state reflects the regular alternation of protein ribbons and zones of bilayer phospholipid separating ribbons, then crystallinity among membranes would provide an independent line of evidence for the widespread occurrence of ribbon structure in membranes. This equation of crystallinity among membranes with the presence of ribbon structure is bolstered by the evidence that cross-linking reagents, such as glutaraldehyde, can convert biological membranes to one gigantic cross-linked macromolecule (Green, Ji & Brucker, 1972). Only extended ribbon structure would allow such complete cross-linking. The purple membrane of halophilic bacteria has been shown to form crystalline arrays characterized by rosettes of three rhodopsin molecules
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(Unwin & Stoeckenius, 1971). This type of crystallinity would be difficult to rationalize in terms of extended ribbon structure. It has to be remembered that the purple membrane is an artificial membrane generated by separating rhodopsin from the rest of the protein components of the bacterial membrane just as the individual complexes of the electron transfer chain can be separated from the other complexes of the mitochondrial inner membrane. We know that the electron transfer chain forms a continuous ribbon when bonded to the tripartite ribbon, but not when separated from the tripartite ribbon (unpublished studies of Haworth). It is thus possible that rhodopsin forms extended ribbon structure when bonded to the tripartite ribbon of the bacterial membrane, but not when detached from the tripartite ribbon, 9. Membrane
Fusion and Ribbon Structure
Some of the most fundamental phenomena of biological membranes (pinocytosis, exocytosis, endocytosis, nuclear pore formation, megamitochondria formation and cell division) depend upon the fusion process (Lucy, 1970; Poole, Howell & Lucy, 1970). The connection between these phenomena and fusion has hitherto been an enigma. It would now appear that the ribbon structure of membranes may be the key to this connection. All the membrane phenomena listed above have one feature in common. A membrane either ruptures to generate two separate membranes, or two separate membranes splice together to form a single continuous membrane. In order for this rupturing and splicing to take place, two membranes have to fuse together a a necessary preliminary. Why is a fusion membrane more susceptible to rupture and splicing than an unfused membrane? In order for a membrane to rupture easily, there has to be a low energy barrier to the transition of phospholipid molecules from the bilayer modality in the intact membrane to the monolayer modality in the ruptured membrane. During rupture of a membrane, two ends are formed and these ends must be covered with a single layer of phospholipid molecules. The thickness of the membrane and the protein-phospholipid interface are the two critical parameters in this bilayer-monolayer transition. The rupture of an unfused membrane will be tenaciously resisted because the paired phospholipid molecules in the bilayer cannot readily rearrange to form a stable monolayer of 50 A in width at each of the two ends formed by rupture (excessive curvature of the interface). When two membranes fuse, the protein-phospholipid interface doubles from 50-60 A to 100-120 A and the curvature of an interface of such length is sufficiently reduced to allow orderly stacking of phospholipid molecules in the monolayer modality. The bilayer-monolayer transition then becomes isoenergetic. For this reason, rupture and splicing
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of membranes are equally feasible after membrane fusion. The point to be emphasized is that the ribbons provide the continuous interface at which this bilayer-monolayer transition can take place and fusion of ribbons reduces the energetic barrier to this transition. 10. The Immune Response and Ribbon Structure The plasma membrane of certain cells (the red blood corpuscle, the lymphocyte and the thymocyte) are highly specialized for the immune response which can take many forms, such as complement fixation, antigeninduced histamine release, polar cap formation, formation of antigenantibody complexes, etc. (Unanue, 1974). The magnitude of the immune response in these cells is sufficiently large that it is necessary to invoke a large number of active centers distributed over the entire surface of the plasma membrane (Unanue, 1974). How can the specialization of the plasma membrane for the immune response be accommodated to the principle of ribbon structure in energy coupling membranes? The answer must be that a new function assumed by a specialized membrane has to be translated by the same structural devices appropriate for translating energy coupling and the control of energy coupling. This would necessarily mean that the plasma membrane of cells specialized for the immune response must contain two kinds of ribbons-one concerned with energy coupling and the other concerned with the immune response. Separate control features would be built into each of these ribbons. The ribbon responsible for the immune response would be segmented like the energy coupling ribbon and each segment would contain the set of proteins required for the full gamut of immune responses. Thus, the immune response, like energy coupling, would be unitized and each membrane would contain ribbons with large numbers of such operational units. Unanue (1974) has estimated that the surface immunoglobulin occupies, at the most, about one-tenth of the total surface membrane of a lymphocyte. If we consider the surface occupied by the immunoglobulin as a rough measure of the surface occupied by the ribbons specialized for the immune response, it follows that a not inconsiderable proportion of the total protein of the membrane (some 20%) is intrinsic to the ribbons committed to the immune response. The immunoglobulin molecules have been shown to be distributed in the form of an irregular network with small microaggregates leaving irregular bare areas of membrane (Unanue, 1974). This type of distribution of immunoglobulin molecules argues for their localization in short strips of immune ribbons-each such ribbon carrying a set of immunoglobulin molecules.
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During polar cap formation, the micro-aggregates of immunoglobulin molecules move together in one end of the cell to form one compact area of coalescence. Two aspects of this translational redistribution of the immunoglobulins in the membrane of the lymphocyte are noteworthy. First, the translation is energy-requiring and is suppressed by reagents that uncouple oxidative phosphorylation such as 2,4-dinitrophenol (Unanue, 1974). This energy-dependence of polar cap formulation emphasizes that the process is a work performance and as such, requires a structural basis. The association of immunoglobulins with ribbon structure could be, in fact, the structural basis for this work performance. Second, the translational process depends not only upon structures intrinsic to the membrane, but also extrinsic to the membrane as can be deduced from the effect of reagents on polar cap formation which specifically suppress the translation properties of these extrinsic structures. This additional evidence of the extensive structural basis of polar cap formation effectively rules out any explanation couched in simplistic terms such as fluid membranes. 11. Intrinsic and Extrinsic Ribbon Structwe The association of extended polymeric arrays of proteins external to the membrane with proteins intrinsic to the membrane has been established for extrinsic arrays such as spectrin, microtubules, microfilaments and actin fibers (Tillack, Marchesi, Marchesi & Steers, 1970; Wang & Richards, 1974; Nicolson & Painter, 1973; Elsgaeter, Sholton & Branton, 1973). It may be possible to visualize such associations in the framework of interconnecting external and internal ribbons. These interconnections would provide the rationalization for phenomena such as polar cap formation in which there is obvious translation of intrinsic membrane proteins from one region of the membrane to the other (Unanue, 1974). The actual translation would be achieved by the external ribbons; the internal ribbons would necessarily move in lock step with the external ribbons by virtue of the interconnections. The point to be emphasized is that there would be no need to invoke translational mobility of the intrinsic membrane proteins to account for such phenomena. The mobile elements would be external to the membrane and these would determine the movement of the internal elements. 12. Summarizing
Comments
The objective of the article was to show how the ribbon structure of membranes provides the link between membrane structure, energy coupling and bioenergetics. Parts of the argument rest on solid experimental evidence.
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A good deal is still based on speculation. But the general thrust of the argument is certainly in the direction of the desired unification. Three central ideas have contributed to this new direction proposed for membranology. The first is the bilayer principle enunciated by J. Danielli some 40 years ago (Danielli & Davson, 1935) and still the cornerstone of all thinking about membrane structure. The second is the concept of intrinsic proteins which are to the protein domain what phospholipids are to the lipid domain (Vanderkooi & Capaldi, 1972). The third is the organization of intrinsic proteins into linear arrays or ribbons-the idea which has been systematically developed in this article. This investigation was supported in part by Program Project Grant GM-12847 of the National Institute of General Medical Sciences. REFERENCES BENEDE~I, E. I. 8c EMMELOT, P. (1965). J. Cell Biol. 26, 299. BLINZENGER, K., REWCASTLE, N. B. & HAGER, H. (1965). J. CeN Biol. 25, 293. BLONDM, G. A. & GREEN, D. E. (1975). Chem. Eng. News 53,26. CHANCE, B. & YGSHIOKA, T. (1966). Arch. Biochem. Biophys. 117, 451. CRANE, F. L., STILES, J. W., PREZBINDOWSKI, F. J., RUJICKA, F. J. & SUN, F. F. (1968). In Regulatory Functions of Biological Membranes, pp. 21-56 (J. Jamefelt, ed.).
Amsterdam: Elsevier Publishing Co. DALES, S. & MOSBACH, E. H. (1969). Virology 35, 564. DANIELLI, J. F. & DAVSON, H. (1935). J. CeN. Physiol. 5, 495. ELSGAETER, A., SHOLTON, D. & BRANTON, D. (1973). J. Cell Biol. 59, 899. FAIN-MAIJREL, M. A. (1968). C. r. hebd. Sednc. Acad. Sci. Paris 267, 1614. FERNANDEZ-MORGAN, H., ODA, T., BLAIR, P. V. & GREEN, D. F. (1964). J. Cell Biol. 22, 63. GIREN, B. B. (1954). Exp. Cell Res. 7, 558. GREEN, D. E. (1972). Ann. N. Y. Acad. Sci. 195, 150. GREEN, D. F. (1974). Ann. N. Y. Acad. Sci. 227, 6. GREEN, D. E. (1974). Biochim. Biophys. Acta 346, 27. GREEN, D. E., BLONDIN, G., KELSSER, R. & SOIJTHARD, J. H. (1975). Proc. nut. Acad. Sci. U.S.A. 72, 896. GREEN, D. E., JI, S. & BRUCKER, R. J. (1972). Bioenergetics 4, 527. GREEN, D. E. & REIBLE, S. (1974). Proc. nut. Acad. Sci. U.S.A. 71, 4850. GREEN, D. E. & REIBLE, S. (1975). Proc. nut. Acad. Sci. U.S.A. 72, 253. HATASE, O., WAKABAYASHI, T., HAYASHI, H. SCGREEN, D. E. (1972). Bbenergetics 3,509. HAYASHI, H., VANDERKOOI, G. & CAPALDI, R. A. (1972). Biochem. Biophys. Res. Commun. 49, 92. IKIMOTO, N., STREETER, F. A., NAKAMURA. A. & GERGELY, J. (1968). J. Ultrastrucf. Res. 23,216. INESI, G. & ASAI, H. (1968). Arch. Biochem. Biophys. 126, 469. KEMENY, G. (1974~). Proc. nat. Acad. Sci. U.S.A. 71, 3064. KEMENY, G. (19746). Proc. nut. Acad. Sci. U.S.A. 71, 3669. KEMENY, G. & SINGER, P. (1975). Proc. nat. Acad. Sci. U.S.A. 72, 2058. KOMAI, H., HIJNTER, D. R. & TAKAHASHI, Y. (1973). Biochem. Biophys. Res. Commun. 53, 182. LAVER, W. J. (1973). Adv. in Virus Res. 18, 57.
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LUCY, J. A. (1970). Nature, Land. 227, 815. MACLENNAN, D. H., OSTWALD, T. J. 8~ STEWART, P. S. (1974). Ann. N. Y. Acad. Sci. 227, 527. MACLENNAN. D. H., TZAGOLOFF, A. & MCCONNELL, D. G. (1967). Blochim. Biophys. Acta 131, 59. MANIUIPP, J., VANDERKOOI, G., HAYASHI, H. & CAPALDI, R. (1973). Biochim. Biophys. Acta 298, 180. NICOLSON, G. L. & PAINTER, R. G. (1973). J. Cell. Biol. 59, 345. O’BR~N, J. S. & SAMPSON, E. L. (1965). J. Lipid Res. 6, 537. ODA, T. &SKI, S. (1966). In Electron Microscopy 1966, vol. 2, pp. 387-388 (R. Uyeda, ed.). Tokyo: Maruzen. OLESZKO, S. & MOUDRIANAKIS, E. N. (1974). J. Cell Biol. 63, 936. POOLE, A. R., HOWELL, J. I. & LUCY, J. A. (1970). Nature, Land. 227, 810. SADLER, M., HUNTER, D. R. & HAWORTH, R. A. (1974). Biochem. Biophys. Res. Commun. 59,804. SOUTHARD, J. H. & GREEN, D. E. (1974). Biochem. Biophys. Res. Commun. 61, 1310. TILLACK, T. W., MARCHESI, S. L., MARCHESI, V. T. & STEERS, E. (1970). Biochim. Biophys. Acta 200, 125. UNANUE, E. R. (1974). Am. J. Path. 77, 2. UNWIN, P. N. T. & STOECKENIUS, W. (1971). Nature New Biol. 233, 152. VANDERKOOI, G. & CAPALDI, R. A. (1972). Ann. N.Y. Acad. Sci. 195, 135. WANG, K. & RICHARDS, F. (1974). J. biol. Chem. 249, 8005.
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The Structure of Biological Membranes in Relation to the Principle of Ehergy Coupling D. E. GREEN Institute for Emzyme Research, University of Wisconsin, Madison, Wisconsin 53706, U.S.A.
The thesis that the structure of biological membranes translates the principle of energy coupling has been systematically developed. The protein ribbon continuum has been identified as the structural feature of membranes which is relevant to this translation. The body of evidence bearing on the widespread occurrence of ribbon structure in membranes has been reviewed. Fusion of membranes, the crystalline state of membranes, as well as the extensive cross-linking of membrane proteins are phenomena predictable from ribbon structure. The possible ways in which ribbon structure in membranes can effectively translate energy coupling, the control of energy coupling, and the immune response have been considered. 1. Introduction
The case for postulating a universal principle of energy that underlies all bioenergetic phenomena has been developed by Green (Green, 1974a,b; Blondin & Green, 1975; Green & Reible, 1974; Green & Reible, 1975; Green, Blondin, Kessler & Southard, 1975) and by Kemeny (Kemeny, 1974u,b; Kemeny & Singer, 1975). In the present article, we shall be considering the implications of this universality of the energy principle for the structure of biological membranes. Since membranes are the instruments of a wide variety of coupled processes (active transport of cations and metabolites, oxidative and photosynthetic phosphorylation, coupled electron flow and hydrolysis of ATP), we may presume that the structure of membranes is appropriate for translating the energy principle that underlies these coupled processes. In fact, we may go further and assert that the struoture of membranes which carry out coupled functions has to embody features which are essential for this translation. Since membrane function is invariably subject to control, the structure of membranes must satisfy two requirements-translation of the function, as well as translation of the control of 271
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this function. This means that all membranes which carry out some form of energy coupling must have structural features in common that are requisite for translating the control and functional parameters of energy coupling. The often repeated cliche that no two membranes are alike should be recognized as a highly dubious assertion, since it denies the limitations on the variability of structure imposed by the requirements for function and control. One must distinguish between variability in respect to details and variability in respect to fundamentals. Certainly there will be variability at the level of chemical and morphological detail, but it is also obvious that variability in respect to the structural features requisite for translating the energy principle and the control of energy coupling will necessarily be severely circumscribed. 2. The Ribbon Structure of the Mitochondrion The structural feature of membranes which we consider most relevant to energy coupling was discovered by Fernandez-Moran, Oda, Blair & Green (1964) in 1961 in their studies of negatively stained mitochondria. They observed paired and closely apposed strips of inner membrane with arrays of headpiece-stalks projecting in one direction on one side of the fused strips and projecting in the opposite direction on the other side. The obvious conclusion to be drawn from these electron micrographs was that the inner membrane had a continuous ribbon structure with headpiece-stalks projecting from the ribbon in precise periodicity. This periodicity showed up sharply when two strips of membrane were fused together-bottom to bottom. Such a simple and obvious interpretation was not drawn, however, because it would have been completely out of line with the then current dogma as to the structure of membranes. Instead, the arrays were considered to be apparent arrays in the same sense that individual tombstones in a cemetery appear as arrays of tombstones when viewed from a sufficient distance. Subsequently, such orderly arrays of projecting headpiece-stalks were found in a wide variety of membranes-sarcoplasmic reticulum (Ikimoto, Streeter, Nakamura & Gergely, 1968; Inesi & Asai, 1968), microsomal membranes (MacLennan, Ostwald & Stewart, 1967), microvilli (Oda & Seki, 1966), chloroplast (Oleszko & Moudrianakis, 1974), plasma membranes (Benedetti & Emmelot, 1965) and viral membranes (Dales & Mosbach, 1968)-and with monotonous regularity, these arrays were rationalized as apparent arrays. F. Crane (Crane, Stiles, Prezbindowski, Rujicka & Sun, 1968) was one of the few to insist on the reality of the continuous array. He produced electron microscopic evidence in negatively stained preparations of mitochondria and submitochondrial particles that
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detergent such as Triton X-100 induced the formation of large numbers of iilamentous structures with arrays of projecting headpiece-stalks. In the study of electron transport particles exposed to the fragmenting action of lysolecithin, Sadler, Hunter & Haworth (1974) found in negatively stained preparations of such treated particles numerous examples of open-ended strips of ribbon structure with arrays of headpiece-stalk projections (see Fig. 1 for a drawing of such structures). In this case, any interpretation in
FIG. 1. A drawing representativeof the ribbon strips observed by Sadler et-al.(1974) in electron micrographs of negatively stained lysolecithin-treated electron transport particles. The cross-section of the ribbon is intended to emphasize the non-membranous character of the ribbon. terms of stacking and apparent arrays could be definitively excluded, since the structures visualized were no longer closed membranes, but fragments of membranes. It was then obvious that a continuous ribbon structure was formed by fragmentation of the inner mitochondrial membrane and that this ribbon structure could not be lipoidal in nature, since lysolecithin effectively eliminated bilayer structure. The continuous, but segmented, structure to which the arrays of headpiece-stalks were attached had to be predominantly protein in nature. The proof of this has been established by direct isolation of the segmented units (Sadler et al., 1974). These continuous structures we have designated as ribbons. The next order of business was to find conditions and reagents which would induce ribbon formation in an across-the-board fashion. The experimental aim was to “ribbonize” the entire inner membrane and to stabilize the membrane in this ribbon&d state. Our first efforts (Haworth and Green, unpublished studies) were directed to a search for conditions which favored this ribbonization of the mitochondrial inner membrane. We found that ribbonization was favored with frozen mitochondria that were thawed under hypotonic conditions at pH 8. To a high degree, such mitochondria
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FIG. 2. A drawing representative of the continuum observed in electron micrographs of negatively stained beef heart mitochondria after freezing, thawing and exposure to alkaline pH (c. 8-O). The drawing of the cross-section of the continuum emphasizes that the structure is a tubular membrane and that there are two ribbons per tubule, separated one from another by a zone of bilayer phospholipid. The electron micrographs from which the drawings were made were prepared by R. A. Haworth.
showed extended ribbons with paired arrays of headpiece-stalks. These ribbons were not membrane fragments, as in the case of lysolecithin-treated particles. They were, in fact, tubular membranes with paired ribbon structure as depicted diagrammatically in Fig. 2. Bilayer phospholipid separated the two ribbons in the tubular membrane. The occurrence of ribbon structure within the intact membrane suggests a possible explanation for the hitherto bizarre observation of cristae showing regular geometrical shape. When the number is two, the tubule is spherical; when the number is three, the tubule is triangular (Blinzenger, Rewcastle & Hagar, 1965); and when the number is four, the tubule has a square crosssectional appearance (Fain-Maurel, 1968). In mitochondria from different sources with tubular cristae, one can readily see these variations in the geometry of the tubular cristae. Once it was recognized that tubularization of the inner membrane is equivalent to ribbonization, some earlier unpublished studies in our laboratory by J. Smoly and T. Wakabayashi came into clearer perspective. These investigators found that silicotungstate induced quantitative tubularization of the inner mitochondrial membrane. The headpiece-stalks in the spherical tubules induced by silicotungstate projected into the interior instead of exteriorily, as in the case of the lysolecithin-induced ribbons. Furthermore, the diameter of the silicotungstate-induced tubules was considerably larger (3-4 times) than the tubules induced by exposure of previously frozen mitochondria to hypotonic alkaline conditions-a token that the number of ribbons per tubule would be at least six.
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The long and short of these experimental studies of Haworth and Green is that they established the reality of ribbon structure in mitochondria and opened the door to phenomena derivative from ribbon structure-phenomena such as tubularization. Apparently, there is a dynamic equilibrium between the planar state of the crista and the tubular&d state in which one crista gives rise to a number of tubules all interconnected. There apparently is also a dynamic equilibrium between extended ribbons and short strips of ribbon. We deduce this from the fact that the ribbons of enormous length seen in tubular cristae of hypotonic mitochondria must have arisen by rearrangement and end-to-end association of shorter structures in the planar membrane from which they are derived. 3. Structural Organization
of the Ribbon Continuum
We may define the ribbon structure of the rnitochondrial inner membrane in the following terms. It is a protein continuum which, like bilayer phospholipid, constitutes a section of the membrane. The inner membrane is thus a composite of a protein domain (the ribbon) and a phospholipid domain (the bilayer zone). The ribbons are separated one from another by a zone of bilayer phospholipid. The cross-sectional diameter of the ribbon, i.e. from one side of the membrane to the other, is about 60-75 A; the comparable dimension for the bilayer is about 50 A. Thus, the protein domain of the membrane is always slightly thicker than the phospholipid domain. The second cross-sectional dimension of the ribbon is about 120 A-wide enough to accommodate a foursome of elongated proteins, each with a diameter of about 30 A. The proteins may extend the width of the membrane (through membrane proteins) or half-way across the membrane (like the phospholipids in the bilayer). The bonds which hold together the proteins in the ribbon structure are predominantly hydrophobic in nature (Komai, Hunter & Takahashi, 1973). Lysolecithin can fragment the segmented ribbon into smaller unitscommonly with two headpiece-stalk projections (Komai et al., 1973). One such unit has a length of about 300 A and in cross-section, the unit would have the geometry of a rectangle 75 x 60 A. The ribbon is apparently formed by the hydrophobic association of linear sets of units, each with two headpiece-stalk projections. 4. Functional Parameters of the Ribbon Continuum The entire energy coupling apparatus of the mitochondrion is localized in the ribbon structure of the inner membrane. This would include the
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systems for active transport of cations, for oxidative phosphorylation, for electron transfer and ATP hydrolysis, and for energized transhydrogenation. We may reasonably assume that mitochondrial coupling functionality is segmented and that each unit of the ribbon with two headpiece-stalk projections has the capacity for all the above-listed coupled functions. Sadler et al. (1974) have shown that lysolecithin strips the electron transfer chain cleanly away from the ribbon structure to which the headpiece-stalk projections are attached, whereas the ribbons in tubularized membranes have their full complement of the electron transfer complexes. These observations suggest that the protein ribbon in the intact inner membrane is a composite of two ribbons-one containing the complexes of the electron transfer chain (the electron transfer chain ribbon) and the other containing the segmented units with headpiece-stalk projections (the tripartite ribbon). These two ribbons would be tightly apposed laterally forming, in effect, a fusion ribbon. The individual ribbons would have a diameter of about 60 A-wide enough to accommodate two elongated proteins, each with a diameter of about 30 A. 5. Translation of Energy Coupling by the Ribbon Continuum Now we may pose the critical question whether ribbon structure has special advantages in respect to the facilitation of energy coupling. We have postulated that the essence of energy coupling is paired charge separation, paired charge movement and paired charge elimination (Blondin & Green, 1975). This requires the parallel and vectorial flow of two charged species, each in a separate reaction center, across the membrane (Blondin 62 Green, 1975). The close apposition of the electron transfer chain ribbon and the tripartite ribbon provides the ideal combination for such paired charge flow (Fig. 3). Since the two moving species must be as close as possible for maximization of coupling, the suitability of the ribbon modality for achieving such close proximity is obvious. This proximity applies not only to the interaction between a moving ion in the electron transfer chain ribbon and a moving ion in the tripartite ribbon, but also to the interaction between paired moving ions, both of which are in the tripartite ribbon. There are two features of ribbon structure that are particularly relevant to the necessities for energy coupling. The first is the straight line geometry of the ribbon in the long dimension and the rectangular geometry in crosssection. For tight apposition of coupled complexes, for minimization of the distance between complexes, and for precise vectorial movements of the two coupled charge species, the geometries inherent in ribbon structure are ideally suited for the requirements of energy coupling. The second feature
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FIG. 3. A drawing illustrating how electron transfer can be.coupled to synthesis of ATP in the ribbon continuum. The flow of the electron in the electron transfer ribbon is vectorially aligned to the flow of the positively charged ionophoric species in the tripartite ribbon. The two ribbons are tightly apposed and in exact register.
has to do with the problem of alignment of complexes in the electron transfer chain ribbon with complexes in the tripartite ribbon. Since the electron transfer chain ribbon has to move with respect to the tripartite ribbon as each electron transfer complex is successively brought into position for coupling with the counterpart complex in the tripartite unit, this would involve the sliding of one ribbon with respect to the other. For such a sliding maneuver, the linear and rectangular geometry of the ribbon would be a great advantage. The advantages of ribbon structure for energy coupling that we have listed should be independent of the length of the ribbon. What, then, would be the advantage of extended ribbon structure for energy coupling? Experiment has shown that the efficiency of energy coupling is greatly reduced when ribbons are fragmented to successively smaller units (Sadler et al., 1974). The 32Pi-ATP exchange activity is an order of magnitude lower on a specific activity basis for the same unit in isolation as compared to the unit which is part of extended ribbon structure of the mitochondrion (Sadler et al., 1974). This observation would suggest that the length of the ribbon may be an important factor in the efficiency of coupling. Chance & Yoshioka (1966) have shown that mitochondria can undergo oscillations in a remarkably regular pattern-oscillations involving the inflow and outflow of ions into the mitochondrion. This regularity bespeaks the synchronizing of metabolic activities within individual mitochondria. It is as if, at a given time, the same complex in each electron transfer chain in a mitochondrion is engaged in coupling. This type of synchronization requires a built-in structural feature and we are proposing that ribbon structure provides this very
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feature. Why synchronization is a desirable property is still an unanswered question, but that it is present in mitochondria is undisputable. During energy coupling, there is interplay between the matrix proteins, the coupling systems of the inner membrane, and the mechanisms for translocation of metabolites. This type of interplay which is part and parcel of synchronization would certainly call for the type of cooperative behavior that only an extended structure like a ribbon should show. 6. Control of Energy Coupling by the Ribbon Continuum The mitochondrion has a control mechanism which regulates the pattern of coupling capability (unpublished studies of Hunter, Haworth and Southard). Mg ‘+ favors, or induces, the state appropriate for oxidative phosphorylation and high-affinity transport of Ca’+ and Mg2+ ; Ca2+ favors, or induces, the state in which the mitochondrion lacks the ability to carry out oxidative phosphorylation, but is capable of low-a6nity active transport of Ca’+, Mg2+ and K+ (Southard & Green, 1974). The Mg2+favored state corresponds to the aggregated configuration, and the Ca2+-favored state to the orthodox configuration of the mitochondrion. In the aggregated configuration, the headpiece-stalk projection appears to be retracted into the membrane; in the orthodox configuration, the headpiece-stalk is known to be extended from the membrane (Hatase, Wakabayashi, Hayashi & Green, 1972). The control mechanism of the mitochondrion very likely controls the contraction and extension of the stalk which thereby controls the movement in and out of the membrane of the headpiece (F,). Concomitant with the contraction and extension of the stalk, there is not only the movement of the headpiece (F,) in and out of the membrane, but also a change in the pattern of energy coupling and a change in the permeability of the membrane to neutral solutes such as sugars. In consequence of these changes which accompany the contraction and extension of the stalk, the cristae undergo a transition from one configurational state to another. The cristae expand and round up when the headpiece is inserted into the membrane (aggregated configuration); and contract and straighten out when the headpiece is ejected from the membrane (orthodox configuration). This type of control mechanism involves a change in state of all the cristae in the mitochondrion. Such a blanket change in the state of the mitochondrial cristae can be most effectively translated by extended ribbon structure. This control mechanism cannot be general for all membranes with projecting headpiece-stalks, since it is only in the mitochondrion, the chloroplast, and bacterial membranes that the ATPase and ATP synthetase are associated
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with the headpiece (ATPase and ATP synthetase are part of the headpiecestalk-basepiece ensemble). In other membranes (the membranes with microheadpiece-stalks), the ATPase is permanently intrinsic to the membrane and is never extruded (MacLennan et al., 1974). The headpiece of the viral membrane has been identsed as a neuramidase (Laver, 1973), the headpiece of the microvilli as a carbohydrate-splitting enzyme (Oda & Seki, 1966). Does the control mechanism in such membranes involve the contraction into, and extension from the membrane of the headpiece? If analogy is to be our guide, it would be predictable that the control mechanism in membranes with projecting microheadpieces would involve the same in-and-out movement of the head pieces and this maneuver would lead to two alternative states of the membrane. In one state, the membrane would have the enzymic capability intrinsic to the headpiece; in the other state, this capability would be absent. The appearance and disappearance of this capability in the membrane would be blanket for the entire organelle. Extended ribbon structure would insure that control would be an across-the-board, rather than a local phenomenon. The proof that headpiece-stalks can contract into the membrane is provided by the evidence of fusion. Two membranes with projecting headpiece-stalks can be induced to undergo fusion (unpublished studies of Wakabayashi and Green). After fusion, the space between the two membranes disappears. If the headpiece-stalk projections were not able to contract into the membrane, fusion of the two membranes would be impossible. It should be recalled that the headpiece-stalks project at least 150 A from the membrane (Fernandez-Moran et al., 1964). Without retraction of the headpieces, the two membranes would never get closer than 300 A apart. The in-and-out movement of the headpieces probably involves the contraction and extension of the stalk. This would mean that the stalk is a contractile protein and that the control maneuver regulates the ion-dependent reversible contraction and relaxation of this protein. Some rearrangement of the proteins in the headpiece would be required for this in-and-out maneuver of the membrane. In the membrane, the headpiece would have to expose a hydrophobic surface. Out of the membrane, the headpiece would have to expose a hydrophilic surface. This change in polarity would necessarily entail some rotation of the proteins in the headpiece. Shape changes in the ribbon continuum go parallel with the changes in configurational state induced by the control mechanism. A variety of membrane phenomena require or depend upon such shape changes. The change in shape of lymphocytes from the stationary to the mobile form is one dramatic example of an energy-requiring control mechanism (Unanue, 1974). The formation of pseudopodia by motile cells is yet another.
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7. How General is Ribbon Structure in Membranes? A crucial question is whether ribbon structure is an intrinsic feature of all membranes, or only of membranes with the capability for active transport or some other form of energy coupling. We are unaware of any biological membrane that shows no evidence of the capacity of energy coupling. The myelin membrane could be considered one such case, but it must be remembered that the myelin membrane is derived from the plasma membrane of the Schwann cell (Giren, 1954) and all plasma membranes unquestionably have the capacity for energy coupling. It is inconceivable that ribbon structure would be intrinsic to the myelin membrane in the early stages, but not in the later stages of differentiation. The relatively low concentration of protein in the myelin membrane (20 % versus 50% or more in other membranes) could be interpreted as a token that the structure of the myelin membrane is atypical (O’Brien 8z Sampson, 1965). The mass ratio of protein ribbon : bilayer phospholipid need not necessarily be 1 : 1 as in the mitochondrion. The zone of bilayer phospholipid in the myelin membrane separating one ribbon from another could be far wider than in other membranes. Hence, a low protein : phospholipid ratio may have no bearing on the presence or absence of ribbon structure. Ribbon structure is compatible with either a low or a high ratio. 8. The Crystalline State of Membranes in Relation to Ribbon Structure
A high proportion of biological membranes have been shown to exist in a checkerboard crystalline state (Green, 1972). This checkerboard crystallinity reflects the precise intercalation of protein and phospholipid domains as Vanderkooi & Copaldi (1972) have shown in two studies of crystalline cytochrome exidase (Maniloff, Vanderkooi, Hayashi & Capaldi, 1973; Hayashi et al., 1972). If, as we suspect, the crystalline state reflects the regular alternation of protein ribbons and zones of bilayer phospholipid separating ribbons, then crystallinity among membranes would provide an independent line of evidence for the widespread occurrence of ribbon structure in membranes. This equation of crystallinity among membranes with the presence of ribbon structure is bolstered by the evidence that cross-linking reagents, such as glutaraldehyde, can convert biological membranes to one gigantic cross-linked macromolecule (Green, Ji & Brucker, 1972). Only extended ribbon structure would allow such complete cross-linking. The purple membrane of halophilic bacteria has been shown to form crystalline arrays characterized by rosettes of three rhodopsin molecules
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(Unwin & Stoeckenius, 1971). This type of crystallinity would be difficult to rationalize in terms of extended ribbon structure. It has to be remembered that the purple membrane is an artificial membrane generated by separating rhodopsin from the rest of the protein components of the bacterial membrane just as the individual complexes of the electron transfer chain can be separated from the other complexes of the mitochondrial inner membrane. We know that the electron transfer chain forms a continuous ribbon when bonded to the tripartite ribbon, but not when separated from the tripartite ribbon (unpublished studies of Haworth). It is thus possible that rhodopsin forms extended ribbon structure when bonded to the tripartite ribbon of the bacterial membrane, but not when detached from the tripartite ribbon, 9. Membrane
Fusion and Ribbon Structure
Some of the most fundamental phenomena of biological membranes (pinocytosis, exocytosis, endocytosis, nuclear pore formation, megamitochondria formation and cell division) depend upon the fusion process (Lucy, 1970; Poole, Howell & Lucy, 1970). The connection between these phenomena and fusion has hitherto been an enigma. It would now appear that the ribbon structure of membranes may be the key to this connection. All the membrane phenomena listed above have one feature in common. A membrane either ruptures to generate two separate membranes, or two separate membranes splice together to form a single continuous membrane. In order for this rupturing and splicing to take place, two membranes have to fuse together a a necessary preliminary. Why is a fusion membrane more susceptible to rupture and splicing than an unfused membrane? In order for a membrane to rupture easily, there has to be a low energy barrier to the transition of phospholipid molecules from the bilayer modality in the intact membrane to the monolayer modality in the ruptured membrane. During rupture of a membrane, two ends are formed and these ends must be covered with a single layer of phospholipid molecules. The thickness of the membrane and the protein-phospholipid interface are the two critical parameters in this bilayer-monolayer transition. The rupture of an unfused membrane will be tenaciously resisted because the paired phospholipid molecules in the bilayer cannot readily rearrange to form a stable monolayer of 50 A in width at each of the two ends formed by rupture (excessive curvature of the interface). When two membranes fuse, the protein-phospholipid interface doubles from 50-60 A to 100-120 A and the curvature of an interface of such length is sufficiently reduced to allow orderly stacking of phospholipid molecules in the monolayer modality. The bilayer-monolayer transition then becomes isoenergetic. For this reason, rupture and splicing
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of membranes are equally feasible after membrane fusion. The point to be emphasized is that the ribbons provide the continuous interface at which this bilayer-monolayer transition can take place and fusion of ribbons reduces the energetic barrier to this transition. 10. The Immune Response and Ribbon Structure The plasma membrane of certain cells (the red blood corpuscle, the lymphocyte and the thymocyte) are highly specialized for the immune response which can take many forms, such as complement fixation, antigeninduced histamine release, polar cap formation, formation of antigenantibody complexes, etc. (Unanue, 1974). The magnitude of the immune response in these cells is sufficiently large that it is necessary to invoke a large number of active centers distributed over the entire surface of the plasma membrane (Unanue, 1974). How can the specialization of the plasma membrane for the immune response be accommodated to the principle of ribbon structure in energy coupling membranes? The answer must be that a new function assumed by a specialized membrane has to be translated by the same structural devices appropriate for translating energy coupling and the control of energy coupling. This would necessarily mean that the plasma membrane of cells specialized for the immune response must contain two kinds of ribbons-one concerned with energy coupling and the other concerned with the immune response. Separate control features would be built into each of these ribbons. The ribbon responsible for the immune response would be segmented like the energy coupling ribbon and each segment would contain the set of proteins required for the full gamut of immune responses. Thus, the immune response, like energy coupling, would be unitized and each membrane would contain ribbons with large numbers of such operational units. Unanue (1974) has estimated that the surface immunoglobulin occupies, at the most, about one-tenth of the total surface membrane of a lymphocyte. If we consider the surface occupied by the immunoglobulin as a rough measure of the surface occupied by the ribbons specialized for the immune response, it follows that a not inconsiderable proportion of the total protein of the membrane (some 20%) is intrinsic to the ribbons committed to the immune response. The immunoglobulin molecules have been shown to be distributed in the form of an irregular network with small microaggregates leaving irregular bare areas of membrane (Unanue, 1974). This type of distribution of immunoglobulin molecules argues for their localization in short strips of immune ribbons-each such ribbon carrying a set of immunoglobulin molecules.
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During polar cap formation, the micro-aggregates of immunoglobulin molecules move together in one end of the cell to form one compact area of coalescence. Two aspects of this translational redistribution of the immunoglobulins in the membrane of the lymphocyte are noteworthy. First, the translation is energy-requiring and is suppressed by reagents that uncouple oxidative phosphorylation such as 2,4-dinitrophenol (Unanue, 1974). This energy-dependence of polar cap formulation emphasizes that the process is a work performance and as such, requires a structural basis. The association of immunoglobulins with ribbon structure could be, in fact, the structural basis for this work performance. Second, the translational process depends not only upon structures intrinsic to the membrane, but also extrinsic to the membrane as can be deduced from the effect of reagents on polar cap formation which specifically suppress the translation properties of these extrinsic structures. This additional evidence of the extensive structural basis of polar cap formation effectively rules out any explanation couched in simplistic terms such as fluid membranes. 11. Intrinsic and Extrinsic Ribbon Structwe The association of extended polymeric arrays of proteins external to the membrane with proteins intrinsic to the membrane has been established for extrinsic arrays such as spectrin, microtubules, microfilaments and actin fibers (Tillack, Marchesi, Marchesi & Steers, 1970; Wang & Richards, 1974; Nicolson & Painter, 1973; Elsgaeter, Sholton & Branton, 1973). It may be possible to visualize such associations in the framework of interconnecting external and internal ribbons. These interconnections would provide the rationalization for phenomena such as polar cap formation in which there is obvious translation of intrinsic membrane proteins from one region of the membrane to the other (Unanue, 1974). The actual translation would be achieved by the external ribbons; the internal ribbons would necessarily move in lock step with the external ribbons by virtue of the interconnections. The point to be emphasized is that there would be no need to invoke translational mobility of the intrinsic membrane proteins to account for such phenomena. The mobile elements would be external to the membrane and these would determine the movement of the internal elements. 12. Summarizing
Comments
The objective of the article was to show how the ribbon structure of membranes provides the link between membrane structure, energy coupling and bioenergetics. Parts of the argument rest on solid experimental evidence.
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A good deal is still based on speculation. But the general thrust of the argument is certainly in the direction of the desired unification. Three central ideas have contributed to this new direction proposed for membranology. The first is the bilayer principle enunciated by J. Danielli some 40 years ago (Danielli & Davson, 1935) and still the cornerstone of all thinking about membrane structure. The second is the concept of intrinsic proteins which are to the protein domain what phospholipids are to the lipid domain (Vanderkooi & Capaldi, 1972). The third is the organization of intrinsic proteins into linear arrays or ribbons-the idea which has been systematically developed in this article. This investigation was supported in part by Program Project Grant GM-12847 of the National Institute of General Medical Sciences. REFERENCES BENEDE~I, E. I. 8c EMMELOT, P. (1965). J. Cell Biol. 26, 299. BLINZENGER, K., REWCASTLE, N. B. & HAGER, H. (1965). J. CeN Biol. 25, 293. BLONDM, G. A. & GREEN, D. E. (1975). Chem. Eng. News 53,26. CHANCE, B. & YGSHIOKA, T. (1966). Arch. Biochem. Biophys. 117, 451. CRANE, F. L., STILES, J. W., PREZBINDOWSKI, F. J., RUJICKA, F. J. & SUN, F. F. (1968). In Regulatory Functions of Biological Membranes, pp. 21-56 (J. Jamefelt, ed.).
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