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Role of lipids in membrane structures
Role of lipids in membrane structures Derek Marsh Max-Planck-lnstitut f/~r biophysikalische Chemie, G6ttingen, Germany Lipids form the basic building blocks of biological membranes but their role in membrane structure is many-fold and can be divided loosely into structural and functional aspects, although evidently both are closely related. This review highlights several key areas: chain interdigitation; domain formation; asymmetry and vesicle transport; modulation of enzyme activity; protein insertion and translocation; lipid-protein interactions; and, lipid diffusion. These are principally those areas that have received special attention during the past year. Current Opinion in Structural Biology 1992, 2:497-502
Introduction The role of lipids in membrane structures is extensive in its scope and heterogeneous in nature. It ranges from simple barrier properties, through the effects of lipid mobility, membrane dynamics and lipid-protein interactions, to protein insertion, endo/exocytosis, domain formation, second-messenger signalling and lipid anchoring of membrane proteins. Some of the latter aspects have been covered by previous and present reports. The consequences on membrane function of lipid mobility alone are many-fold, as this forms the molecular basis of all membrane dynamic processes (reviewed in [1]). Such processes include respiratory-chain electron transport, ligand-induced protein aggregation, receptor-mediated endocytosis, intracellular membrane trafficking and recycling, cell movement, and developmental changes. In view of the compositional heterogeneity of biological membranes, it is not surprising that, in reviewing the field, much information of direct relevance continues to come from the study of defined lipid model systems, in addition to the results obtained on natural membranes. The novel results obtained with pure lipid systems will point the way and stimulate future attempts to identi~y such processes also in native membranes. The present review focuses on those aspects of the role of lipids in membrane structures that have received special attention in publications during the previous year, and which can be divided roughly into those affecting membrane structure and those influencing membrane function. Direct lipid properties are dealt with first, followed by the effects of lipids on membrane function.
Lipid chain interdigitation Interdigitation of the lipid chains across the bilayer leaflet might constitute a possible means of communication
across the membrane. Such a mechanism could play a significant role in the case of sphingolipids, for which the naturally occurring species can have rather large chain asymmetries. The fact that, in general, membrane lipids contain chains of unequal length is likely also to be an important structural feature in most biological membranes. The problem of chain asymmetry has been approached by biophysical techniques in a series of systematic studies with synthetic phospholipids (for a review, see [2]). In the latest of these investigations, asymmetry between the sn-1 and sn-2 chains in saturated diacyl phosphatidylcholines (PtdCho)has been varied at constant mean chain length [3",4"']. Such extensive comparisons of the thermotropic transitions and phase-separation characteristics of members of homologous lipid series are helping to delineate in much greater detail the nature of lipid chain interdigitation. The type of chain interdigitation is determined by the degree of chain-length asymmetry, and the resulting chain-end perturbations are reflected in a diagnostic fashion by the thermodynamic properties of the lipid bilayer assemblies (see Fig. 1). Pronounced effects on lipid mixing properties are also observed, depending on the difference in chain asymmetry between the component lipids.
Lipid domains One of the potentially important features of membrane organization is the segregation of functionally differentiated in-plane domains. Recently, lipid domains have been visualized in erythrocyte membranes by fluorescence microscopy [5"*]. This is highly significant, as most experiments demonstrating domain formation have been performed with model membranes composed of lipid mixtures. Particularly interesting in the latter area is the connectivity between domains in two-phase binary lipid mixtures that has been determined by photobleach-
Abbreviations PtdCho---phosphati~lylcholine; Ptdlns--phosphatidylinositol; PldGro~phosphatidylglycerol; Pldl-ll~phosphatidic acid. (~ Current Biology Ltd ISSN 0959-440X
497
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~O
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/ ~
D / C(10)'C(22) FtdCho
/c,~0,:c( 12)
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Fig. 1. Dependence of the chain-melting
C(19):C(13)F~dCho
transition temperature, Tin, on the normalized difference in length, AC/CL, between the sn-1 and sn-2 chains of phosphatidylcholines (PtdCho) with a mean chain length of 16 (solid line) and 17 (dashed line) carbon atoms. Models indicating the mode of chain interdigitation and chain-end perturbations (indicated by the wavy line) in the gel phase are given above each region. Published with permission [4".].
I
C(12~C(20) \ PtdCflo
i
C)
i
PtdCho
I
ing techniques [6°-]. A complete fluorescence recovery is only obtained with labelled lipid molecules that are in domains connected over distances of the order of the size of the bleaching spot ( ,-, 1 I.tm). The point at which connectivity of fluid domains is achieved is accompanied by an abrupt increase in the fluorescence recovery. Clearly, the issue of connectivity is related quite generally to the reactivity of membrane components within domains, for example in photosynthetic electron transfer [7]. Novel methods, including neutron scattering, for deter mining phase diagrams of binary lipid mixtures, and some possible functional consequences, have also been reviewed recently [8]. Techniques for detecting fluidphase domain formation by lipid crosslinking have been presented for lipid model membranes [9] and point the way to further studies on more complex membrane sys terns.
Lipid asymmetry and intracellular vesicle transport The well documented asymmetry in phospholipid distribution between the opposite faces of many biological membranes (see [10]) has been demonstrated to be of functional significance by the discovery of a specific ATPdependent translocase for amino-phospholipids. This area has been excellently reviewed by Devaux [11.,],
whose group was responsible for the original identification of the phospholipid flippase activity in erythrocyte membranes. In natural membranes, transbilayer movement of phospholipids can be relatively rapid, compared with the transbilayer exchange in lipid bilayer model membranes, which is extremely slow (for example, see [12]). (In contrast, neutral lipids such as protonated fatty acids, cholesterol and diacylglycerols [13"] can flip-flop rapidly.) Most interesting is the in vivo functional role of phospholipid asymmetry, one plausible candidate for which may be the maintenance of membrane shape and control of vesiculation, as demonstrated by recent model experiments [14"] and theoretical calculations [15..]. In addition, lipid asymmetry has been shown to affect the transbilayer movement of platelet-activating factor, and was suggested to be implicated in its release [16]. A related finding of extreme relevance in cellular systems is that the distribution of phosphatidylinositol (Ptdlns) may be central to the control of the vesiculation necessary for intracellular membrane trafficking from the Golgi (reviewed in [17"]). The distribution of phosphoinositides is determined by the Ptdlns-transfer protein [18-], whose critical importance is emphasized by the recent identification of this protein with the SEC14p protein of the secretory pathway in yeast [19]. The hypothesis is that the SEC14p (Ptdlns-transfer) protein is responsible for elevating the Ptdlns: PtdCho ratio in the Golgi membranes, which in turn leads to a stimulation of secretion (see Fig. 2). A possible mechanism for budding of vesicles from the trans-Golgi cisternae may involve the accu-
Role of lipids in membrane structures Marsh 499 mulation of highly charged polyphosphoinositides which are synthesized from PtdIns within the Golgi itself.
Enzyme activation and modulation of activity by lipids One functional role of membrane lipids is in the activation of membrane-associated proteins. The lipiddependent regulatory enzyme protein kinase C provides a good example of this. The Ca 2 + -dependent binding of protein kinase C to negatively charged lipids, which is essential for the enzyme activation, has been shown to induce quantitative and reversible clustering of phosphatidic acid (PtdH) and phosphatidylglycerol (PtdGro) in mixed PtdCho membranes [20°o]. A similar clustering of negatively charged lipids was also found to be induced by two other proteins of molecular weight 64 and 32 kD purified from brain. Other classes of membrane-associated Ca 2 + -binding proteins were shown not to have this property, suggesting that the formation of segregated domains of anionic phospholipids is necessary for the function of protein kinase C, and may contribute unique features to the total calcium response of the cell. Conformational changes in cytochrome c upon binding to negatively charged lipid membranes have been detected by resonance Raman spectroscopy [21..], so demonstrating an explicit molecular mechanism for the activation of membrane proteins by lipids. One of the membrane bound conformations involves the opening of the haem crevice, which results in a shift of redox potential from + 0.02V to - 0 . 3 1 / - 0.41 V and establishes an equilibrium between a six-coordinate low-spin to a five-coordinate high-spin configuration of the prosthetic group, which themselves have different redox potentials. The conformational equilibrium of the bound protein was found to be sensitive both to lipid composition and phase state. In particular, quite low concentrations of diacTlglycerol were found to have marked conformational effects ([21"]; see also [13 °] for interfacial effects of diacylglycerol). The accompanying changes observed in the lipid membrane curvature may provide a general. mechanism for activation of membrane-bound proteins, and in particular of protein kinase C. The activity of integral membrane enzymes and transport proteins also can be modulated by lipid composition. For the Ca 2 + -ATPase from sarcoplasmic reticulum, an opti-
Golgi (inactive)
mum chain length of C(18:1) to C(20:1) has been found for supporting activity of the enzyme reconstituted in dimonounsaturated PtdChos [22,23]. [C(n:m) indicates a fatty acyl chain of n carbon atoms in length that contains m double bonds.] Progress is now being made towards defining this lipid-dependent modulation in terms of the elementary steps in the overall reaction cycle. In contrast to the native enzyme or reconstitutions in lipids of longer chain length, the stoichiometry of Ca 2 + binding is not 2:1 but 1:1 for the enzyme in diC(14:l)PtdCho [24]. Recent kinetic studies have found a lower rate of phosphoenzyme formation in diC(14:l)PtdCho relative to native membranes, which was attributed to a reduction in rate of conformational change following ATP binding [25, ]. The association of different phospholipids with the ATPase therefore affects the conformational flexibility of this membrane-bound enzyme. Rod outer segment disc membranes from vertebrate retina are characterized by a high proportion of polyunsaturated lipids, principally those containing docosahexaenoic acid (C22:6). Although photochemical and biochemical functionality has been demonstrated for rhodopsin reconstituted in diC(14:0)PtdCho, a phospholipid with saturated chains [26o], it is clear that the longer polyunsaturated lipid chains have a profound effect on the overall visual transduction cycle. Recent experiments on reconstituted systems have shown a steep increase in the equilibrium ratio of metarhodopsin II to metarhodopsin I following bleaching which is associated with an increased degree of unsaturation in the lipid chains [27oo]. A possible reason for the unique properties of the polyunsaturated lipids is suggested by the reduced cohesion between their chains, as evidenced by the unusually low lateral elastic constants for diC(20:4)PtdCho bilayers [28] and the lack of interaction of polyunsaturated lipids with cholesterol [29]. The molecular basis for these chain-chain interactions has been investigated recently by means of 2H NMR spectroscopy [30"].
Protein insertion and translocation Although the membrane insertion or transmembrane transport of many newly synthesized proteins involves specific protein receptor or recognition systems, it seems inevitable that, in nearly all cases involving membrane proteins, the lipid component of the membrane phase
Golgi (active)
> Ptdlns/PtdCho,l,
Ptdlns/PtdChoT SEC14p
,.@ @
Fig. 2. The Ptdlns:PtdCho ratio hypothesis (Ptdlns, phosphatidylinositol; PtdCho, phosphatidylcholine). The Ptdlnstransfer activity of the SEC14p protein is responsible for elevating (T) the Ptdlns:PtdCho ratio of yeast Golgi membranes above that of the bulk membranes (endoplasmic reticulum and plasma membranes), leading to activation of secretion. Adapted from [17"*].
500 Lipids must play a part. In the special case of apocytochrome c, which bears no leader sequence, no such receptor has been identified, and evidence is steadily accumulating on the way in which import into the mitochondrion may take place by a mechanism involving the lipids of the outer mitochondrial membrane [31",32,33]. Progress is also being made in defining the way in which synthetic protein signal sequences interact with lipid bilayer membranes, as a model for the role of lipids in the total insertion process, which in some cases is mediated by a transmembrane potential [34",35]. A series of experiments of considerable significance [36"] have demonstrated the requirement of negatively charged phospholipids for the translocation of an outer membrane precursor protein across the inner membrane of Escherichia coll. In cells genetically manipulated to interrupt specifically the pathway for synthesis of PtdGro, it was found that export of newly synthesized prePhoE could be restored by addition of PtdGro, diphosphatidylglycerol, PtdH, PtdIns or phosphatidylserine, so demonstrating the participation of anionic phospholipids in the protein translocation process. In complementary experiments with model membranes, it was shown that negatively charged lipids are essential for ~z-helix formation in the signal peptide of prePhoE, which was proposed to be the functional conformation during protein translocation [37o].
Fig. 3. Steric exclusion between the myelin basic protein (MBP) and the myelin proteolipid protein (PLP) in double recombinants with dimyristoyl phosphatidylglycerol (PtdGro) at a PtdGro: PLP ratio of nt. A critical lower PtdGro: PLP ratio, Nc, is required such that binding of MBP to the lipid is undisturbed by the PLP. Where n t < Nc, MBP is unable to bind to the first shell of lipids, N1, surrounding the PLP [39.]. Published with permission [39"].
Lipid-protein interactions
example, the outer membrane proteins of E. coli, which are thought to possess lB-barrel structures.
Studies on lipid-protein interactions inevitably continue to be of fundamental importance to considerations of the role of lipids in membrane structure. This area was reviewed in this journal last year [38]. Recent developments include the extension of studies on reconstituted systems involving a single peripheral or integral membrane protein to double reconstitutions with both types of protein [39"]. It was found that there was no specific association between the two major proteins of myelin in the reconstituted membrane; rather, the interaction was one of steric repulsion in which the integral proteolipid protein excluded the peripheral basic protein from binding to anionic lipids in its immediate boundary shell (see Fig. 3). Mutual perturbations of the lipid interactions with one protein by the other protein were observed, indicating communication between the two proteins via the lipid phase.
From the point of view of the functional implications of lipid interactions with integral proteins, a direct association with the intramembranous surface of the acetylcholine receptor protein has been demonstrated for the class of aminated local anaesthetic molecules that are known to be non-competitive blockers of the agonistevoked response [41].
The effects of protein secondary structure on lipid interactions with integral proteins have been studied for the M13 bacteriophage coat protein in its a-helical and polymeric IB-sheet forms [40.]. The rates of lipid exchange at the intramembranous surface of the protein aggregates in lipid bilayers were found to be slower, and the lipid se lectivities were found to be greater, for the IS-sheet than for the cz-helical form of the protein. These differences are attributable, at least in part, to the more extended nature of the [~-sheet structure for the same number of residues in the hydrophobic span. It remains to be seen to what extent these results may be extrapolated to, for
Lipid diffusion and electron transport The role of ubiquinone as a mobile carrier in mitochondrial electron transport has long been suggested, and its diffusion can constitute the rate-limiting step in the inner membrane (for example, see/42]). Such arguments have been based in part on diffusion measurements with lipid analogues in mitochondrial membranes. Recently, the rates of diffusion of ubiquinone have been measured in model membranes and found to be similar to those of the bilayer lipids [43.,44]. These and previous measurements on lipid-supplemented mitochondrial membranes [45] help to establish the diffusional and collisional nature of electron transport between different complexes of the respiratory chain, emphasizing the functional involvement of the lipid phase in the inner mitochondrial membrane. The role of restricted diffusion of the corre sponding carrier lipid, plastoquinone, in photosynthetic systems has been reviewed recently [7].
Role of lipids in membrane structures Marsh 501 Conclusion The field o f lipid function in m e m b r a n e structures is multi-faceted and virtually unlimited in scope. It can be anticipated that this will continue to be an area o f intense research and that many new insights will be obtained in this complex area in the years to come.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest ** of outstanding interest 1.
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2.
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3. .
LtN I-IN, WANG ZQ, HUANGC: The Influence of Acyl Chainlength Asymmetry on the Phase Transition Parameters of Phosphatidylcholine Dispersions. Biochim Biophys Acta 1991, 1067:17-28. Thermodynamic paranleters of chain melting are determined for two series of PtdChos with extensive ranges of chain asymmetries and mean chain lengths of 14 and 15 carbon atoms. 4. .•
BULTMANNT, LtN HN, WANG ZQ, HUANG C: Thermotropic and Mixing Behaviour of Mixed-chain Phosphatidylcholines with Molecular Weights Identical with That of L-~x-Dipalmitoylphosphatidylcholine. Biochemistry 1991, 30:7194-7202. A systematic study of the effects of chain asymmetry on the lipid chainmelting transition and on mixing properties in binary systems. RODGERSW, GLASER M: Characterization of Lipid Domains in Erythrocyte Membranes. Proc Natl Acad Sci USA 1991, 88:1364-1368. A direct demonstration of the existence of large lipid domains in a biological membrane. 5. .•
6. ..
BULTMANNT, VAZ WLC, MELO EEC, StSK RB, THOMPSON TE: Fluid-phase Connectivity and Translational Diffusion in a Eutectic, Two-component, Two-phase Phosphatidylcholtne Bilayer. Biochemistry 1991, 30:5573-5579. A determination of the connectivity between domains in laterally phaseseparated lipid mixtures. 7.
LAVERGNE J, JOLIOT P: Restricted Diffusion in Photosynthetic Membranes. Trends Biochem Sci 1991, 16:129-134.
8.
KNOLLW, SCHMIDT G, ROTZER H, HENKEL T, PFEIFFER W, SACKMANNE, MITIZER-NEHERS, SPINKEJ: Lateral Order in Binary Lipid Alloys and its Coupling to Membrane Functions. Chem Phys L~oids 1991, 57:363-374.
9.
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10.
Diacylglycerols in PhosphoUpid BRayers, J Biol Chem 1991, 266:1177-1186. The mode of intercalation and interfacial conformation of diacylglycerol is investigated by NMR, and rapid transbilayer movement is demonstrated. 14. •
FARGEE, DEVAUXPF: Shape Changes of Giant Liposomes Induced by an Asymmetric Transmembrane Distribution of Phospholipids. Biophys J 1992, 61:347-357. Model experiments illustrate a possible functional role for lipid asymmetry in biological membranes. 15. LIPOWSKYPc The Conformation of Membranes. Nature 1991, ** 349:475-481. An accessible review of current theoretical thinking on factors affecting shape, adhesion, fluctuations, etc. of membranes. 16.
BRATrONDL, KAILEYJM, CLAYKL, HENSON PM: A Model for the Extracellular Release of PAF - - the Influence of Plasma Membrane Phospholipid Asymmetry. Biochim Biophys Acta 1991, 1062:24-34.
17. CLEVESA, MCGEE T, BANKAITISV: Phospholipid Transfer Pro** teins: a Biological Debut. Trends Cell Biol 1991, 1:30-34. Proposals for the mechanism whereby the SEC14p (Ptdlns-transfer) protein may activate secretion from the Golgi in yeast, and a review of the involvement of phospholipid-transfer proteins in phospholipid equilibration and in secretion. 18. WIRTZ KW& Phospholipid Transfer Proteins. Annu Rev • Biochem 1991, 60:73-99. An up-m-date review of the properties and possible functions of phospholipid-transfer proteins. 19.
BANKAnaSVA, AITKENJR, CLEVESAE, DOWHANW: An Essential Role for Phospholipid Transfer Protein in Yeast Golgi Function. Nature 1990, 347:561-562.
20. **
BAZI MID, NEtSESTUEN GL: Extensive Segregation of Acidic Phospholipids in Membranes Induced by Protein Kinase-C and Related Proteins. Biochemistry 1991, 30:7961-7969. The authors demonstrate the formation of anionic lipid-rich domains upon activation by the Ca2+ -dependent binding of protein kinase C to mixed lipid membranes. 21. ..
HEIMBURGT, HILDEBRANDTP, MARSH D: Cytochrome c-Lipid Interactions Studied by Resonance Raman and 3lp NMR Spectroscopy--Correlation Between the Conformational Changes of the Protein and the Lipid Bilayer. Biochemistry 1991, 30:9084-9089. A direct demonstration of conformational changes in cytochrome c upon binding to negatively charged lipid bilayers and their modulation by lipid state and composition. 22.
CAFFREYM, FEIGENSON GW: Fluorescence Quenching in Model Membranes. 3. Relationship Between Calcium Adenosinetriphosphatase Enzyme Activity and the Affinity of the Protein for Phosphatidylcholines with Different Acyl Chain Characteristics. Biochemistry 1981, 20:1949--1961.
23.
HULLINF, BOSSANTMJ, SALEMN: Aminophospholipid Molecular Species Asymmetry in the Human Erythrocyte Plasma Membrane. Biochim Biophys Acta 1991, 1061:15-25.
JOHANNSSON A, KEIGHTLEY CA~ SMITH GA, PdCHARDS CD, HESKETH TR, METCALFEJC: The Effect of BRayer Thickness and n-Alkanes on the Activity of the (Ca2+ +Mg2+) dependent ATPase of Sarcoplasmic Reticulum. J Biol Chem 1981, 256:1643-1650.
24.
11. DEVAUXPF: Static and Dynamic Asymmetry in CeR Mem** branes. Biochemistry 1991, 30:1163-1173. A review of current knowledge regarding lipid translocase activities and asymmetry.
MICHELANGELIF, ORLOWSKIS, CHAMPEILP, GRIMESEA, EASTJM, LEE AG: Effects of Phospholipids on Binding of Calcium to (CaX+-Mg2+)-ATPase. Biochemistry 1990, 29:8307-8312.
25. •
12.
13. •
Wt~LEYWC, THOMPSON TE: Transbilayer and Interbilayer Phospholipid Exchange in Dimyristoylphosphatidytcholinedimyristoylphosphatidylethanolamine Large UnRamellar Vesicles. Biochemistry 1991, 30:1702-1709. HAMILTONJA, BHAMIDIP#TI SP, KODALI DR, SMALLDM: The Interfacial Conformation and Transbilayer Movement of
MICHELANGELIF, GRIMES EA, EAST JM, LEE AG: Effects of Phospholipids on the Function of (Ca2+-Mg2+)-ATPase. Biochemistry 1991, 30:342-351. A detailed kinetic study of the effec~ of a short-chain lipid in modulating the ATPase activity. 26. •
MITCHELLDC, KIBELBEK J, LITMAN BJ: Rhodopsin in Oimyristoylphosphatidylcholine-reconstituted Bilayers Forms Metarhodopsin II and Activates Gt. Btodxrmistry 1991, 30:37-42.
502
Lipids Demonstration of the photochemical and functional activity of rhodopsin in a saturated, short-chain phospholipid: significant because of the large number of biophysical studies conducted on such systems. MITCHELL DC, STRAtlME M, LrrMAN BJ: Role of sn-1Saturated, sn-2-polyunsaturated Phospholipids in Control of Membrane Receptor Conformational Equilibration: Effects of Cholesterol and Acyl Chain Unsaturation on the Metarhodopsin I-Metarhodopsin II Equilibrium. BiD chemistry 1992, 31:662-670. A demonstration of the pronounced effect of lipid chain polyunsaturation on the meta I-meta II equilibrium of rhodopsin, and the modulation of this equilibration in a compensatow fashion by cholesterol and temperature. This paper also includes a discussion of the sn-1/sn-2 chain asymmetry in membrane lipids in general. 27. o•
KUSTERSR, DOWHAN W, DE KRUIJFF B: Negatively-charged Phospholipids Restore prePhoE Translocation Across Phosphatidylglycerol-depleted Escherichia coli Inner Membranes. J Biol Chem 1991, 266:8659-8662. A demonstration of the direci~ involvement of negatively charged lipids in the translocation of an outer-membrane protein across the E. coli plasma membrane. 36. •e
37. •
KELLERRCA, KILLIANJA, DE KRUIJFFB: Anionic Phospholipids Are Essential for c~-Helix Formation of the Signal Peptide of prePhoE on Interaction with Phospholipid Vesicles. Bio chemistry 1992, 31:1672-1677. The formation of tz-helical structure is emerging as a motif for interaction of signal sequences with anionic lipids and, in this case, appears to correlate with translocation competency.
28.
NEEDHAMD, NUNN RS: Elastic Deformation and Failure of Lipid Bilayer Membranes Containing Cholesterol. Biophys J 1990, 58:997-1009.
38.
29.
KARIELN, DAVIDSON E, KEOUGH ~ : Cholesterol Does Not Remove the Gel-Liquid Crystalline Phase Transition of Phosphatidylcholines Containing Two Polyenoic Chains. Biochim Biophys Acta 1991, 1062:70-76.
•
30. •
BARRYJA, TROUARD TP, SALMON A, BROWN MF: Low-ternperature 2H NMR Spectroscopy of Phospholipld Bilayers Containing Docosahexaenoyl (22 : 6o3) Chains. Biochemistry 1991, 30:8386-8394. The polyunsaturated chain occurring predominantly in rod outer segment disc membranes does not alter appreciably the overall s n l chain order of mixed-chain PtdChos in the fluid phase, but does promote ordered chain packing in the gel phase. The chain-melting temperature, however, lies below the physiological range. 31. •
DE JONGH HHJ, KILLIANJA, DE KRUIJFF B: A Water-Lipid Interface Induces a Highly Dynamic Folded State in Apocytochrome c and Cytochrome c, which May Represent a Common Folding Intermediate. Biochemistry 1992, 31:1636-1643. Micelle-association induces a high degree of secondary structure in apocytochrome c, but the tertiary folding is labile for both apo- and holoproteins. 32.
33.
39.
SANKARAMMB, BROPHY PJ, MARSH D: Lipid-Protein and Protein-protein Interactions in Double Recombinants of Myelin Proteolipid Protein and Myelin Basic Protein with Dimyristoylphosphatidylglycerol. Biochemistry 1991, 30:5866-5873. Mutual interactions of a peripheral and an integral membrane protein reconstituted with negatively charged lipids are shown to arise from steric exclusion. 40. •
PEELENSJCJ, SANDERSJC, HEMMINGAMA, MARSHD: Stoichiometry, Selectivity, and Exchange Dynamics of Lipid-Protein Interaction with Bacteriophage M13 Coat Protein Studied by Spin Label Electron Spin Resonance. Effects of Protein Secondary Structure. Biochemistry 1992, 31:2670-2677. Experiments and discussion on the effects of protein secondary structure on lipid interactions with integral proteins. 41.
HORVATHLI, ARIASHR, HANKOVSZKYHO, HIDEG K, BARRANTES VJ, MARSHD: Association of Spin-labeled Local Anaesthetics at the Hydrophobic Surface of Acetylcholine Receptor in Native Membranes from Torpedo m a r m o r a t g Biochemistry 1990, 29:8707-8713.
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D Marsh, Max-Planck-lnstitut ffir biophysikalische Chemie, Abteilung Spektroskopie, WD-3400 G6ttingen, Germany.
Introduction The role of lipids in membrane structures is extensive in its scope and heterogeneous in nature. It ranges from simple barrier properties, through the effects of lipid mobility, membrane dynamics and lipid-protein interactions, to protein insertion, endo/exocytosis, domain formation, second-messenger signalling and lipid anchoring of membrane proteins. Some of the latter aspects have been covered by previous and present reports. The consequences on membrane function of lipid mobility alone are many-fold, as this forms the molecular basis of all membrane dynamic processes (reviewed in [1]). Such processes include respiratory-chain electron transport, ligand-induced protein aggregation, receptor-mediated endocytosis, intracellular membrane trafficking and recycling, cell movement, and developmental changes. In view of the compositional heterogeneity of biological membranes, it is not surprising that, in reviewing the field, much information of direct relevance continues to come from the study of defined lipid model systems, in addition to the results obtained on natural membranes. The novel results obtained with pure lipid systems will point the way and stimulate future attempts to identi~y such processes also in native membranes. The present review focuses on those aspects of the role of lipids in membrane structures that have received special attention in publications during the previous year, and which can be divided roughly into those affecting membrane structure and those influencing membrane function. Direct lipid properties are dealt with first, followed by the effects of lipids on membrane function.
Lipid chain interdigitation Interdigitation of the lipid chains across the bilayer leaflet might constitute a possible means of communication
across the membrane. Such a mechanism could play a significant role in the case of sphingolipids, for which the naturally occurring species can have rather large chain asymmetries. The fact that, in general, membrane lipids contain chains of unequal length is likely also to be an important structural feature in most biological membranes. The problem of chain asymmetry has been approached by biophysical techniques in a series of systematic studies with synthetic phospholipids (for a review, see [2]). In the latest of these investigations, asymmetry between the sn-1 and sn-2 chains in saturated diacyl phosphatidylcholines (PtdCho)has been varied at constant mean chain length [3",4"']. Such extensive comparisons of the thermotropic transitions and phase-separation characteristics of members of homologous lipid series are helping to delineate in much greater detail the nature of lipid chain interdigitation. The type of chain interdigitation is determined by the degree of chain-length asymmetry, and the resulting chain-end perturbations are reflected in a diagnostic fashion by the thermodynamic properties of the lipid bilayer assemblies (see Fig. 1). Pronounced effects on lipid mixing properties are also observed, depending on the difference in chain asymmetry between the component lipids.
Lipid domains One of the potentially important features of membrane organization is the segregation of functionally differentiated in-plane domains. Recently, lipid domains have been visualized in erythrocyte membranes by fluorescence microscopy [5"*]. This is highly significant, as most experiments demonstrating domain formation have been performed with model membranes composed of lipid mixtures. Particularly interesting in the latter area is the connectivity between domains in two-phase binary lipid mixtures that has been determined by photobleach-
Abbreviations PtdCho---phosphati~lylcholine; Ptdlns--phosphatidylinositol; PldGro~phosphatidylglycerol; Pldl-ll~phosphatidic acid. (~ Current Biology Ltd ISSN 0959-440X
497
4~PtS Lipids
0-. 0 O
~O
C(16):C(16) PldCho
.-(,~
PtdChotdCho
,
~
,
x
~*'x
T
•
,
O "x
',O
c04;:C(m) N.~ PidCho PidCho ~ C(17):C(15) ~ Pd l ChoPtdCho ~ X X
"
/
x ~
• ,. ~, 0
o\
C(13):C(19) \
etdCho
~
T
j' " ~,~
(~ z ,
,o
,"
/ ~
D / C(10)'C(22) FtdCho
/c,~0,:c( 12)
~
~,
~c~o
/C(1 I):C(21)
Fig. 1. Dependence of the chain-melting
C(19):C(13)F~dCho
transition temperature, Tin, on the normalized difference in length, AC/CL, between the sn-1 and sn-2 chains of phosphatidylcholines (PtdCho) with a mean chain length of 16 (solid line) and 17 (dashed line) carbon atoms. Models indicating the mode of chain interdigitation and chain-end perturbations (indicated by the wavy line) in the gel phase are given above each region. Published with permission [4".].
I
C(12~C(20) \ PtdCflo
i
C)
i
PtdCho
I
ing techniques [6°-]. A complete fluorescence recovery is only obtained with labelled lipid molecules that are in domains connected over distances of the order of the size of the bleaching spot ( ,-, 1 I.tm). The point at which connectivity of fluid domains is achieved is accompanied by an abrupt increase in the fluorescence recovery. Clearly, the issue of connectivity is related quite generally to the reactivity of membrane components within domains, for example in photosynthetic electron transfer [7]. Novel methods, including neutron scattering, for deter mining phase diagrams of binary lipid mixtures, and some possible functional consequences, have also been reviewed recently [8]. Techniques for detecting fluidphase domain formation by lipid crosslinking have been presented for lipid model membranes [9] and point the way to further studies on more complex membrane sys terns.
Lipid asymmetry and intracellular vesicle transport The well documented asymmetry in phospholipid distribution between the opposite faces of many biological membranes (see [10]) has been demonstrated to be of functional significance by the discovery of a specific ATPdependent translocase for amino-phospholipids. This area has been excellently reviewed by Devaux [11.,],
whose group was responsible for the original identification of the phospholipid flippase activity in erythrocyte membranes. In natural membranes, transbilayer movement of phospholipids can be relatively rapid, compared with the transbilayer exchange in lipid bilayer model membranes, which is extremely slow (for example, see [12]). (In contrast, neutral lipids such as protonated fatty acids, cholesterol and diacylglycerols [13"] can flip-flop rapidly.) Most interesting is the in vivo functional role of phospholipid asymmetry, one plausible candidate for which may be the maintenance of membrane shape and control of vesiculation, as demonstrated by recent model experiments [14"] and theoretical calculations [15..]. In addition, lipid asymmetry has been shown to affect the transbilayer movement of platelet-activating factor, and was suggested to be implicated in its release [16]. A related finding of extreme relevance in cellular systems is that the distribution of phosphatidylinositol (Ptdlns) may be central to the control of the vesiculation necessary for intracellular membrane trafficking from the Golgi (reviewed in [17"]). The distribution of phosphoinositides is determined by the Ptdlns-transfer protein [18-], whose critical importance is emphasized by the recent identification of this protein with the SEC14p protein of the secretory pathway in yeast [19]. The hypothesis is that the SEC14p (Ptdlns-transfer) protein is responsible for elevating the Ptdlns: PtdCho ratio in the Golgi membranes, which in turn leads to a stimulation of secretion (see Fig. 2). A possible mechanism for budding of vesicles from the trans-Golgi cisternae may involve the accu-
Role of lipids in membrane structures Marsh 499 mulation of highly charged polyphosphoinositides which are synthesized from PtdIns within the Golgi itself.
Enzyme activation and modulation of activity by lipids One functional role of membrane lipids is in the activation of membrane-associated proteins. The lipiddependent regulatory enzyme protein kinase C provides a good example of this. The Ca 2 + -dependent binding of protein kinase C to negatively charged lipids, which is essential for the enzyme activation, has been shown to induce quantitative and reversible clustering of phosphatidic acid (PtdH) and phosphatidylglycerol (PtdGro) in mixed PtdCho membranes [20°o]. A similar clustering of negatively charged lipids was also found to be induced by two other proteins of molecular weight 64 and 32 kD purified from brain. Other classes of membrane-associated Ca 2 + -binding proteins were shown not to have this property, suggesting that the formation of segregated domains of anionic phospholipids is necessary for the function of protein kinase C, and may contribute unique features to the total calcium response of the cell. Conformational changes in cytochrome c upon binding to negatively charged lipid membranes have been detected by resonance Raman spectroscopy [21..], so demonstrating an explicit molecular mechanism for the activation of membrane proteins by lipids. One of the membrane bound conformations involves the opening of the haem crevice, which results in a shift of redox potential from + 0.02V to - 0 . 3 1 / - 0.41 V and establishes an equilibrium between a six-coordinate low-spin to a five-coordinate high-spin configuration of the prosthetic group, which themselves have different redox potentials. The conformational equilibrium of the bound protein was found to be sensitive both to lipid composition and phase state. In particular, quite low concentrations of diacTlglycerol were found to have marked conformational effects ([21"]; see also [13 °] for interfacial effects of diacylglycerol). The accompanying changes observed in the lipid membrane curvature may provide a general. mechanism for activation of membrane-bound proteins, and in particular of protein kinase C. The activity of integral membrane enzymes and transport proteins also can be modulated by lipid composition. For the Ca 2 + -ATPase from sarcoplasmic reticulum, an opti-
Golgi (inactive)
mum chain length of C(18:1) to C(20:1) has been found for supporting activity of the enzyme reconstituted in dimonounsaturated PtdChos [22,23]. [C(n:m) indicates a fatty acyl chain of n carbon atoms in length that contains m double bonds.] Progress is now being made towards defining this lipid-dependent modulation in terms of the elementary steps in the overall reaction cycle. In contrast to the native enzyme or reconstitutions in lipids of longer chain length, the stoichiometry of Ca 2 + binding is not 2:1 but 1:1 for the enzyme in diC(14:l)PtdCho [24]. Recent kinetic studies have found a lower rate of phosphoenzyme formation in diC(14:l)PtdCho relative to native membranes, which was attributed to a reduction in rate of conformational change following ATP binding [25, ]. The association of different phospholipids with the ATPase therefore affects the conformational flexibility of this membrane-bound enzyme. Rod outer segment disc membranes from vertebrate retina are characterized by a high proportion of polyunsaturated lipids, principally those containing docosahexaenoic acid (C22:6). Although photochemical and biochemical functionality has been demonstrated for rhodopsin reconstituted in diC(14:0)PtdCho, a phospholipid with saturated chains [26o], it is clear that the longer polyunsaturated lipid chains have a profound effect on the overall visual transduction cycle. Recent experiments on reconstituted systems have shown a steep increase in the equilibrium ratio of metarhodopsin II to metarhodopsin I following bleaching which is associated with an increased degree of unsaturation in the lipid chains [27oo]. A possible reason for the unique properties of the polyunsaturated lipids is suggested by the reduced cohesion between their chains, as evidenced by the unusually low lateral elastic constants for diC(20:4)PtdCho bilayers [28] and the lack of interaction of polyunsaturated lipids with cholesterol [29]. The molecular basis for these chain-chain interactions has been investigated recently by means of 2H NMR spectroscopy [30"].
Protein insertion and translocation Although the membrane insertion or transmembrane transport of many newly synthesized proteins involves specific protein receptor or recognition systems, it seems inevitable that, in nearly all cases involving membrane proteins, the lipid component of the membrane phase
Golgi (active)
> Ptdlns/PtdCho,l,
Ptdlns/PtdChoT SEC14p
,.@ @
Fig. 2. The Ptdlns:PtdCho ratio hypothesis (Ptdlns, phosphatidylinositol; PtdCho, phosphatidylcholine). The Ptdlnstransfer activity of the SEC14p protein is responsible for elevating (T) the Ptdlns:PtdCho ratio of yeast Golgi membranes above that of the bulk membranes (endoplasmic reticulum and plasma membranes), leading to activation of secretion. Adapted from [17"*].
500 Lipids must play a part. In the special case of apocytochrome c, which bears no leader sequence, no such receptor has been identified, and evidence is steadily accumulating on the way in which import into the mitochondrion may take place by a mechanism involving the lipids of the outer mitochondrial membrane [31",32,33]. Progress is also being made in defining the way in which synthetic protein signal sequences interact with lipid bilayer membranes, as a model for the role of lipids in the total insertion process, which in some cases is mediated by a transmembrane potential [34",35]. A series of experiments of considerable significance [36"] have demonstrated the requirement of negatively charged phospholipids for the translocation of an outer membrane precursor protein across the inner membrane of Escherichia coll. In cells genetically manipulated to interrupt specifically the pathway for synthesis of PtdGro, it was found that export of newly synthesized prePhoE could be restored by addition of PtdGro, diphosphatidylglycerol, PtdH, PtdIns or phosphatidylserine, so demonstrating the participation of anionic phospholipids in the protein translocation process. In complementary experiments with model membranes, it was shown that negatively charged lipids are essential for ~z-helix formation in the signal peptide of prePhoE, which was proposed to be the functional conformation during protein translocation [37o].
Fig. 3. Steric exclusion between the myelin basic protein (MBP) and the myelin proteolipid protein (PLP) in double recombinants with dimyristoyl phosphatidylglycerol (PtdGro) at a PtdGro: PLP ratio of nt. A critical lower PtdGro: PLP ratio, Nc, is required such that binding of MBP to the lipid is undisturbed by the PLP. Where n t < Nc, MBP is unable to bind to the first shell of lipids, N1, surrounding the PLP [39.]. Published with permission [39"].
Lipid-protein interactions
example, the outer membrane proteins of E. coli, which are thought to possess lB-barrel structures.
Studies on lipid-protein interactions inevitably continue to be of fundamental importance to considerations of the role of lipids in membrane structure. This area was reviewed in this journal last year [38]. Recent developments include the extension of studies on reconstituted systems involving a single peripheral or integral membrane protein to double reconstitutions with both types of protein [39"]. It was found that there was no specific association between the two major proteins of myelin in the reconstituted membrane; rather, the interaction was one of steric repulsion in which the integral proteolipid protein excluded the peripheral basic protein from binding to anionic lipids in its immediate boundary shell (see Fig. 3). Mutual perturbations of the lipid interactions with one protein by the other protein were observed, indicating communication between the two proteins via the lipid phase.
From the point of view of the functional implications of lipid interactions with integral proteins, a direct association with the intramembranous surface of the acetylcholine receptor protein has been demonstrated for the class of aminated local anaesthetic molecules that are known to be non-competitive blockers of the agonistevoked response [41].
The effects of protein secondary structure on lipid interactions with integral proteins have been studied for the M13 bacteriophage coat protein in its a-helical and polymeric IB-sheet forms [40.]. The rates of lipid exchange at the intramembranous surface of the protein aggregates in lipid bilayers were found to be slower, and the lipid se lectivities were found to be greater, for the IS-sheet than for the cz-helical form of the protein. These differences are attributable, at least in part, to the more extended nature of the [~-sheet structure for the same number of residues in the hydrophobic span. It remains to be seen to what extent these results may be extrapolated to, for
Lipid diffusion and electron transport The role of ubiquinone as a mobile carrier in mitochondrial electron transport has long been suggested, and its diffusion can constitute the rate-limiting step in the inner membrane (for example, see/42]). Such arguments have been based in part on diffusion measurements with lipid analogues in mitochondrial membranes. Recently, the rates of diffusion of ubiquinone have been measured in model membranes and found to be similar to those of the bilayer lipids [43.,44]. These and previous measurements on lipid-supplemented mitochondrial membranes [45] help to establish the diffusional and collisional nature of electron transport between different complexes of the respiratory chain, emphasizing the functional involvement of the lipid phase in the inner mitochondrial membrane. The role of restricted diffusion of the corre sponding carrier lipid, plastoquinone, in photosynthetic systems has been reviewed recently [7].
Role of lipids in membrane structures Marsh 501 Conclusion The field o f lipid function in m e m b r a n e structures is multi-faceted and virtually unlimited in scope. It can be anticipated that this will continue to be an area o f intense research and that many new insights will be obtained in this complex area in the years to come.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest ** of outstanding interest 1.
KNOWLESPF, MARSHD: Magnetic Resonance of Membranes, Biochem J 1991, 274:625-641.
2.
HUANGC: Mixed-chain Phospholipids and lnterdigitated Bilayer Systems, Klin Wocbenschr 1991, 68:149-165.
3. .
LtN I-IN, WANG ZQ, HUANGC: The Influence of Acyl Chainlength Asymmetry on the Phase Transition Parameters of Phosphatidylcholine Dispersions. Biochim Biophys Acta 1991, 1067:17-28. Thermodynamic paranleters of chain melting are determined for two series of PtdChos with extensive ranges of chain asymmetries and mean chain lengths of 14 and 15 carbon atoms. 4. .•
BULTMANNT, LtN HN, WANG ZQ, HUANG C: Thermotropic and Mixing Behaviour of Mixed-chain Phosphatidylcholines with Molecular Weights Identical with That of L-~x-Dipalmitoylphosphatidylcholine. Biochemistry 1991, 30:7194-7202. A systematic study of the effects of chain asymmetry on the lipid chainmelting transition and on mixing properties in binary systems. RODGERSW, GLASER M: Characterization of Lipid Domains in Erythrocyte Membranes. Proc Natl Acad Sci USA 1991, 88:1364-1368. A direct demonstration of the existence of large lipid domains in a biological membrane. 5. .•
6. ..
BULTMANNT, VAZ WLC, MELO EEC, StSK RB, THOMPSON TE: Fluid-phase Connectivity and Translational Diffusion in a Eutectic, Two-component, Two-phase Phosphatidylcholtne Bilayer. Biochemistry 1991, 30:5573-5579. A determination of the connectivity between domains in laterally phaseseparated lipid mixtures. 7.
LAVERGNE J, JOLIOT P: Restricted Diffusion in Photosynthetic Membranes. Trends Biochem Sci 1991, 16:129-134.
8.
KNOLLW, SCHMIDT G, ROTZER H, HENKEL T, PFEIFFER W, SACKMANNE, MITIZER-NEHERS, SPINKEJ: Lateral Order in Binary Lipid Alloys and its Coupling to Membrane Functions. Chem Phys L~oids 1991, 57:363-374.
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Diacylglycerols in PhosphoUpid BRayers, J Biol Chem 1991, 266:1177-1186. The mode of intercalation and interfacial conformation of diacylglycerol is investigated by NMR, and rapid transbilayer movement is demonstrated. 14. •
FARGEE, DEVAUXPF: Shape Changes of Giant Liposomes Induced by an Asymmetric Transmembrane Distribution of Phospholipids. Biophys J 1992, 61:347-357. Model experiments illustrate a possible functional role for lipid asymmetry in biological membranes. 15. LIPOWSKYPc The Conformation of Membranes. Nature 1991, ** 349:475-481. An accessible review of current theoretical thinking on factors affecting shape, adhesion, fluctuations, etc. of membranes. 16.
BRATrONDL, KAILEYJM, CLAYKL, HENSON PM: A Model for the Extracellular Release of PAF - - the Influence of Plasma Membrane Phospholipid Asymmetry. Biochim Biophys Acta 1991, 1062:24-34.
17. CLEVESA, MCGEE T, BANKAITISV: Phospholipid Transfer Pro** teins: a Biological Debut. Trends Cell Biol 1991, 1:30-34. Proposals for the mechanism whereby the SEC14p (Ptdlns-transfer) protein may activate secretion from the Golgi in yeast, and a review of the involvement of phospholipid-transfer proteins in phospholipid equilibration and in secretion. 18. WIRTZ KW& Phospholipid Transfer Proteins. Annu Rev • Biochem 1991, 60:73-99. An up-m-date review of the properties and possible functions of phospholipid-transfer proteins. 19.
BANKAnaSVA, AITKENJR, CLEVESAE, DOWHANW: An Essential Role for Phospholipid Transfer Protein in Yeast Golgi Function. Nature 1990, 347:561-562.
20. **
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HULLINF, BOSSANTMJ, SALEMN: Aminophospholipid Molecular Species Asymmetry in the Human Erythrocyte Plasma Membrane. Biochim Biophys Acta 1991, 1061:15-25.
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24.
11. DEVAUXPF: Static and Dynamic Asymmetry in CeR Mem** branes. Biochemistry 1991, 30:1163-1173. A review of current knowledge regarding lipid translocase activities and asymmetry.
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25. •
12.
13. •
Wt~LEYWC, THOMPSON TE: Transbilayer and Interbilayer Phospholipid Exchange in Dimyristoylphosphatidytcholinedimyristoylphosphatidylethanolamine Large UnRamellar Vesicles. Biochemistry 1991, 30:1702-1709. HAMILTONJA, BHAMIDIP#TI SP, KODALI DR, SMALLDM: The Interfacial Conformation and Transbilayer Movement of
MICHELANGELIF, GRIMES EA, EAST JM, LEE AG: Effects of Phospholipids on the Function of (Ca2+-Mg2+)-ATPase. Biochemistry 1991, 30:342-351. A detailed kinetic study of the effec~ of a short-chain lipid in modulating the ATPase activity. 26. •
MITCHELLDC, KIBELBEK J, LITMAN BJ: Rhodopsin in Oimyristoylphosphatidylcholine-reconstituted Bilayers Forms Metarhodopsin II and Activates Gt. Btodxrmistry 1991, 30:37-42.
502
Lipids Demonstration of the photochemical and functional activity of rhodopsin in a saturated, short-chain phospholipid: significant because of the large number of biophysical studies conducted on such systems. MITCHELL DC, STRAtlME M, LrrMAN BJ: Role of sn-1Saturated, sn-2-polyunsaturated Phospholipids in Control of Membrane Receptor Conformational Equilibration: Effects of Cholesterol and Acyl Chain Unsaturation on the Metarhodopsin I-Metarhodopsin II Equilibrium. BiD chemistry 1992, 31:662-670. A demonstration of the pronounced effect of lipid chain polyunsaturation on the meta I-meta II equilibrium of rhodopsin, and the modulation of this equilibration in a compensatow fashion by cholesterol and temperature. This paper also includes a discussion of the sn-1/sn-2 chain asymmetry in membrane lipids in general. 27. o•
KUSTERSR, DOWHAN W, DE KRUIJFF B: Negatively-charged Phospholipids Restore prePhoE Translocation Across Phosphatidylglycerol-depleted Escherichia coli Inner Membranes. J Biol Chem 1991, 266:8659-8662. A demonstration of the direci~ involvement of negatively charged lipids in the translocation of an outer-membrane protein across the E. coli plasma membrane. 36. •e
37. •
KELLERRCA, KILLIANJA, DE KRUIJFFB: Anionic Phospholipids Are Essential for c~-Helix Formation of the Signal Peptide of prePhoE on Interaction with Phospholipid Vesicles. Bio chemistry 1992, 31:1672-1677. The formation of tz-helical structure is emerging as a motif for interaction of signal sequences with anionic lipids and, in this case, appears to correlate with translocation competency.
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29.
KARIELN, DAVIDSON E, KEOUGH ~ : Cholesterol Does Not Remove the Gel-Liquid Crystalline Phase Transition of Phosphatidylcholines Containing Two Polyenoic Chains. Biochim Biophys Acta 1991, 1062:70-76.
•
30. •
BARRYJA, TROUARD TP, SALMON A, BROWN MF: Low-ternperature 2H NMR Spectroscopy of Phospholipld Bilayers Containing Docosahexaenoyl (22 : 6o3) Chains. Biochemistry 1991, 30:8386-8394. The polyunsaturated chain occurring predominantly in rod outer segment disc membranes does not alter appreciably the overall s n l chain order of mixed-chain PtdChos in the fluid phase, but does promote ordered chain packing in the gel phase. The chain-melting temperature, however, lies below the physiological range. 31. •
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D Marsh, Max-Planck-lnstitut ffir biophysikalische Chemie, Abteilung Spektroskopie, WD-3400 G6ttingen, Germany.