Department of Biochemistry, State University of New York at Buffalo, Buffalo, New York 14214, USA
ABSTRACT
Recent studies of structure-function relationships in
biological membranes have revealed fundamental con-
cepts concerning the regulation of cellular membrane
function by membrane lipids. Considerable progress
has been made in understanding the roles played by
two membrane lipids: cholesterol and phosphatidyl-
ethanolamine. Cholesterol has been shown to regulate
ion pumps, which in some cases show an absolute de-
pendence on cholesterol for activity. These studies sug-
gest that an essential role that cholesterol plays inmammalian cell biology is to enable crucial membraneenzymes to provide function necessary for cell survival.
Studies of phosphatidylethanolamine regulation of
membrane protein activity and regulation of mem-brane morphology led to hypotheses concerning theroles for this particular lipid in biological membranes.
New information on lipid-protein interactions and on
the nature of the lipid head groups has permitted thedevelopment of mechanistic hypotheses for the regula-
tion of membrane protein activity by phosphatidyl-ethanolamine. In addition, intermediates in the lamellar-
nonlamellar phase transitions of membrane systems
containing phosphatidylethanolamine, or other lipids
with similar properties, have recently been implicated
in facilitating membrane fusion. Finally, studies of
transmembrane movement of lipids have provided newinsight into the regulation of membrane lipid asym-
metry and the biogenesis of cell membranes. These
kinds of studies are harbingers of a new generation of
progress in the field of cell membranes.-YEAGLE,
P. L. Lipid regulation of cell membrane structure and
function. FASEBJ. 3: 1833-1842; 1989.
Key Words: lipid bilayer . hydrophobic region . membrane
cholesterol calcium pump hexagonal (II) phase head
group protein . cell membrane fusion
THE STUDY OF CELL MEMBRANES, their structure andfunction, is a relatively new field. Although a numberof important contributions date back to the early daysof this century, intense activity and progress in this fieldis marked primarily from the period of the late 1960s
and early 1970s. Only recently has a clear picture of cellmembrane structure and function emerged.
The major features of cell membrane structure thatwe know to be important to their function in living or-ganisms have been described in detail. For example, allcell membranes contain a lipid bilayer. The structure ofthe lipid bilayer is determined in large part by thehydrophobic effect (which also controls protein struc-ture). In particular, it is the repulsion of the lipidhydrocarbon chains by the water structure that drivesthese chains into an environment sequestered fromwater. The amphipathic structure of the polar mem-brane lipids then directly determines the bilayer struc-ture by providing a hydrophobic environment in themiddle of the bilayer for the hydrocarbon chains, withthe lipid polar head groups encountering the aqueousphase.
Cell membranes usually work best when the lipid bi-layer is in the liquid crystalline state. As indicated bythe term “liquid crystal’ the interior of a lipid bilayeris distinctly different from liquid hydrocarbon, althoughboth are hydrophobic. Dynamically, the lipid bilayer ishighly anisotropic; much of the interior of a bilayer iswell ordered, and only a small region in the middle isliquid-like. The conformation of the lipid hydrocarbonchains (as well as the conformation of many of the lipidhead groups) in the bilayer are well described.
All cell membranes contain protein. The membraneproteins may be integrated into the lipid bilayer or maysimply be associated with the membrane. When themembrane proteins are integrated into the bilayer, thetransmembrane portions of the protein consist pre-dominantly of hydrophobic amino acids, making thetransmembrane segment compatible with the hydro-phobic interior of the lipid bilayer. These transmem-brane segments may adopt the a-helical conformation.At the end of these hydrophobic transmembrane regions,a relatively high incidence of charged amino acids isoften found. As a result, the transmembrane proteinsare firmly locked in their position in the lipid bilayer bythe hydrophobic effect; the charged regions cannotenter the hydrophobic interior of the membrane andthe hydrophobic portions of the protein are incompati-ble with water.
Lipids and proteins can diffuse in the plane of themembrane. This may involve relatively free diffusionover long distances, or the membrane proteins may be
I
1834 Vol. 3 May 1989 The FASEB Journal YEAGLE
limited to a finite region of the membrane in whichdiffusion can occur. Alternatively, some plasma mem-brane proteins may be anchored to the membrane skel-eton and show little capability of lateral diffusion.
Early in the modern period of membrane studies, thefluid-mosaic model was presented to describe a struc-ture that was at once dynamic yet ordered (1). This wasan important hypothesis that directed much work in thefield. More recently we have come to understand insome depth just what this dynamic yet ordered mem-brane structure is.
Most cell membranes are not well described by the il-lustrations commonly used to represent membranestructure. The protein content is sufficiently high inmost cellular membranes so that the membrane surfacearea occupied by the membrane proteins is as extensiveor more so as the surface area occupied by the lipids inthe bilayer. It is estimated that in many cellular mem-branes only about three layers of lipid separate theproteins at the point of closest approach of nonag-gregated membrane proteins. Therefore, most draw-ings of biological membranes show too much lipidbilayer. Figure 1 is an attempt to schematicallydescribe a more accurate relationship between the in-tegral membrane proteins and the lipids in a cell mem-brane. Both a hypothetical side view and top view arepresented.
Figure 1. Schematic representation of the relationship between the
amount of lipid bilayer and the amount of protein in a typical cellu-
lar membrane. Top shows the side view and the bottom a surfaceview of the membrane. The membrane lipids are represented by the
balls with two chains attached to each and the large Structures are
the proteins. The inner mitochondrial membrane would have an
even higher protein content and thus less lipid bilayer than thisfigure represents. The myelin membrane would have a lower pro-
tein content and more lipid bilayer than represented in this figure.
These and other structural features of cell mem-branes have been elaborated over the past 2 decades ofresearch (further reading and references in thesegeneral areas can be found in ref 2). What is now com-ing into its own is the study of the mechanism by whichstructural elements of a cell membrane are exploited toregulate the function of that cell membrane.
The following discussion concentrates on several rep-resentative areas of investigation in which recentprogress has been made in uncovering regulatory rela-tionships between cell membrane structure and cellmembrane function. In karticular, regulation of mem-brane function by mefrtbrane lipids is an area of intensecurrent investigation and will be emphasized in thisreview. For clarity, specific examples will be used to II-lustrate each point rather than a comprehensive com-pendium of all the papers published on each subject.
LIPID REGULATION OF MEMBRANE
PROTEIN FUNCTION
Lipids and proteins are known to coexist as closelypacked neighbors in cell membranes. The lipid compo-sition of most cell membranes is complex and fairlytightly regulated metabolically. In this context, onemight expect cell membrane lipids to play a role in cellmembrane protein activity. It has not proved easy toexplore this hypothesis, however. Membrane-boundenzymes have extensive hydrophobic regions andusually require a lipid bilayer to maintain activity. Insome cases, detergent micelles can substitute for thelipid bilayer, protecting the aqueous media from con-tacting the hydrophobic transmembrane region of themembrane proteins. Therefore, separation of the mem-brane enzymes from their native environment to iden-tify the details of the lipid requirements for an enzymeis difficult and has hindered progress in this field. As aresult, an absolute requirement for a particular lipid tosupport the activity of a membrane-bound enzyme hasbeen difficult to document. A notable exception is /3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) for whichan absolute requirement for the choline head group hasbeen described (3). In this section, recent progress inunderstanding the modulation of membrane proteinsby two membrane lipids, cholesterol and phosphatidyl-ethanolamine, will be discussed.
Cholesterol modulation of ion pumps
Cholesterol modulation of two ion pumps has beenstudied in detail by several groups of workers: Na,K-ATPase and Ca2-ATPase. The Na,K-ATPase ofplasma membranes is the enzyme responsible forpumping sodium out of the cell and potassium into thecell against their respective concentration gradients. Inthe erythrocyte membrane, the ratio is 3:2 (Na4/K), sothat the pump is electrogenic. These properties placethis enzyme in a central role in a number of cellularprocesses, including sodium cotransport systems andthe establishment of electrical potentials along theplasma membrane.
REGULATION OF CELL MEMBRANES 1835
Several groups have studied cholesterol modulationof the enzyme from the human erythrocyte. Early re-constitution experiments hoted an inhibition of theenzyme by cholesterol (4). Subsequently, studies ofNaF,K+ATPase in human erythrocyte membranesshowed inhibition of activity by high levels of mem-
brane cholesterol (5, 6).These results were echoed in other membranes. For
example, in rabbit erythrocyte membranes (7), guineapig erythrocyte membranes (8), rat liver membranes(9), and kidney basolateral membranes (10), high cho-lesterol levels (above those found in the native mem-branes) inhibit the ouabain-sensitive ATP hydrolyzingactivity.
This inhibition probably resUlts from the generalphysical effects of cholesterol on a membrane. Choles-terol leads to an increase in the anisotropic motional or-dering of the lipid bilayer of the membrane due to theeffects of its rigid sterol structure on the lipid compo-nents of the membranes (11). This general increase inordering may also lead to an increase in the orderingof the conformation of the Na,K-ATPase. A reductionin the capability of the Na,K-ATPase to undergo con-formational transitions would thereby inhibit its func-tion, perhaps by inhibiting the E, to E2 conformationalchange suggested to be integral to its catalytic cycle.
Cholesterol, however, has another remarkable effecton the Na,K-ATPase (10). When low levels of cho-lesterol are present in the membrane, cholesterol stimu-lates the enzyme. Stimulation is not readily explainedby a bulk cholesterol effect on the membrane lipidproperties, because it is difficult to postulate that inhibi-tion of conformational transitions of the protein mightboth inhibit and stimulate the enzyme. Furthermore,the stimulation was shown to be structurally specific:lanosterol was less capable than cholesterol of stimulat-ing the enzyme and ergosterol had virtually no capabil-ity to stimulate the enzyme. This stimulatory effect isbest explained by a direct sterol-protein interaction,with a site (or sites) on the enzyme that would providethe structural specificity and the mechanism for stimu-lation of the enzyme activity. Further experimentationis needed to test this hypothesis.
The acetylcholine receptor is another example of amembrane protein that appears to require cholesterolto function properly (12).
These results may provide a clue to the essential roleof cholesterol in mammalian cells, which is that cho-lesterol is required for certain important membranefunctions. This essential role of cholesterol has beenfound at the cellular level (13). For example, cells witha requiremhnt for exogenous cholesterol need at leastsmall amounts of,the necessary sterol to exhibit growth.Although other sterols may operate synergistically athigher sterol contents, removal of the specific sterolfrom the media inhibits growth. Other sterols cannotsubstitute for this obligatory requirement at low sterolcontent.
Therefore, it appears that recent evidence supportsthe hypothesis that cholesterol-requiring cells (such asmammalian cells) need cholesterol to maintain the ac-
tivity of enzymes (such as the Na,K-ATPase men-tioned above, or protein kinase) (13) crucial to thegrowth and development of the cell. The way in whichthe cholesterol requirement is manifest might bethrough cholesterol-protein interactions, which aremediated by cholesterol-specific sites on the cholesterol-sensitive proteins.
The calcium pump provides another interesting ex-ample of cholesterol modulation of membrane-boundenzyme activity. The calcium pump in question is theCa2-ATPase of the rabbit fast twitch muscle sarco-plasmic reticulum. This enzyme has been observed tooptimally pump two calcium ions per ATP hydrolyzedand can maintain transmembrane gradients of three orfour orders of magnitude in ion concentration. This en-zyme has been extensively studied not only for its ownintrinsic role in muscle contraction, but also as a moregeneric example of an ion pump because of the successof investigators in a number of laboratories in recon-stituting the activity of this enzyme in bilayers ofdefined lipid composition.
Early data suggested that the level of cholesterol inthe membrane did not affect the activity of the calciumpump. Although this conclusion was questioned, subse-quent work supported the conclusion that the calciumpump protein is not sensitive to cholesterol. The mech-anism by which the calcium pump was rendered im-
mune to the presence of cholesterol was suggested to bethe exclusion of cholesterol from the immediate vicinity(lipid annulus) of the protein (14). This suggestion is adirect manifestation of the hypothesis that lipid effectson membrane proteins are mediated through directbinding to the protein of the lipid in question.
In reconstitution experiments in which phosphatidyl-ethanolamine (PE)’ was used as a dominant lipid com-ponent in the membrane, cholesterol appeared tostimulate the calcium pump (15). Thus, under specialcircumstances, cholesterol appears capable of stimulat-ing function of the calcium pump protein. It is notknown at this time whether cholesterol interacts with orbinds to the calcium pump protein under these condi-tions of reconstitution. Therefore, the hypothesis out-lined above requires further testing.
PE regulation of membrane protein activity
Phospholipids as well as cholesterol are capable ofregulating the activity of membrane proteins. One sys-tem, which has been studied by several groups withconsiderable agreement in results, is the regulation ofthe calcium pump protein by PE.
The first report of an effect of PE on calcium pump
activity was published in 197 about a reconstitutedsystem containing soy PE and egg phosphatidylcholine
(PC) (16). At low to moderate PE levels, PE appearedto stimulate the calcium pump. However, important in-formation about the Structure of the reconstituted sys-tem and its lipid composition was not available then.Subsequent studies (17) have shown that importantnonbilayer structures could form from membranes con-taining the lipid components used in the 1975 reconsti-tution studies. In particular, a substantial region of thephase diagram of this lipid system represents hexagonalII phase, and other regions contain nonbilayer isotropicstructures. Therefore, it was suggested that thesechanges in structure should be considered in relation tothe noted effects of PE on the calcium pump protein.
More recently, two studies again pointed to the roleof PE in stimulating the calcium pump protein. In onestudy, the PE content of sarcoplasmic reticulum mem-branes was altered by chemically labeling the PE headgroups (18). This chemical alteration led to a decreasein calcium pump activity. To the extent that this loss offunction resulted from the reduction in PE (and notfrom the introduction of the chemical label), this studyimplied a role of PE in the native membrane on cal-cium pump function.
In the second study, reconstituted membranes wereused, and the increase in pump activity with increasein PE content was once again demonstrated, althoughactual PE content of the reconstituted membranes wasnot reported (19).
What mechanism might be operating to promote cal-cium pump function in the presence of PE in the mem-brane? The suggestion that it was a preferential andspecific interaction between PE and the calcium pumpprotein was challenged by the finding that monogalac-tosyldiglyceride (MGDG) also stimulated the pump ac-tivity, analogous to PE (19).
One property that PE and MGDG have in commonis the ability to form the hexagonal (II) phase. There-fore, it was necessary to investigate whether the forma-tion of hexagonal (II) phase was important to thestimulation of the calcium pump. Figure 2 shows anexample of such a study in which the calcium pumpwas reconstituted into membranes containing variouslevels of PE. Two different PEs were used. One wouldundergo the transition to the hexagonal (II) phase underthe conditions of the experiments and the other wouldnot. Both PEs stimulated the calcium pump. However,the loss of transport activity when one of the systemslost its bilayer structure was apparent. Therefore, thestimulation appears unrelated to the formation of thehexagonal (II) phase.
It is not clear why PE is capable of significant stimu-lation of the calcium pump protein. One possibilitythat remains to be investigated is the role of the bilayersurface in controlling membrane protein function. Thesurface of PE bilayers is distinctly different than that ofmany other phospholipids. The PE surface is poorlyhydrated and tends to interact with other surfaces,whether they are on other bilayers or proteins, ratherthan interact directly with the aqueous phase (seebelow) (20). The possibility of interactions between thebilayer surface and the extramembranous portions of
PC/(PC+PE)
Figure 2. Calcium uptake in reconstituted membrane vesicles con-taining Ca-ATPase from rabbit muscle sarcoplasmic reticulum as afunction of the PE content of the vesicles. Plot of the percentage ofrecovery of calcium uptake at 37#{176}Cas a function of the PC/(PC +PE)molar ratio for vesicles reconstituted with transphosphatidylated(from egg PC) PE/egg PC (El) or soybean PE/egg pc (U) lipid mix-tures. Because of the greater level of unsaturation in the soy PE,lipid mixtures with that lipid will undergo the lamellar-to-hexagonal (II) phase transition more readily. At 75% PE content
and higher, the predominant form is the isotropic structures or thehexagonal (II) phase, and the vesicles can no longer trap calcium.In the other reconstituted membranes, the system remains lamellar
throughout and the monotonic increase in the stimulation by PE isapparent for all levels of PE content. (From K. -H. Cheng, S. W.Hui, and P. L. Yeagle, unpublished results.)
the calcium pump protein should be examined as a pos-sible mechanism for lipid regulation of this enzyme.
LIPID-PROTEIN INTERACTIONS
It is likely that the interaction between lipids and pro-teins in membranes is one of the mechanisms for theregulation of membrane protein function by mem-brane phospholipids. This interaction might involve:1) a specific binding of the lipid to sites on the protein;2) a more general, nonspecific interaction such as hasbeen embodied in the term “lipid annulus”; or 3) asurface-surface interaction involving the surface of thebilayer and the extramembranous portion of theprotein.
The first two concepts have been extensively studied.The following will review the current state of this field.
Do membrane lipids bind to membrane proteins?
Observations have been reported that suggest thatmembrane lipids bind to membrane proteins. For ex-ample, glycophorin from the human erythrocyte mem-brane is isolated with tightly bound lipids that cannotbe removed without extreme conditions. The lipidsbound to glycophorin appear to be enriched in thephosphatidylinositols (21). Cytochrome oxidase (EC1.9.3.1) from mitochondria is another example in whichtightly bound lipids that cannot be easily removed arefound with the protein after isolation. In this case the
RECULATION OF CELL MEMBRANES 1837
bound lipids are enriched in diphosphatidylglycerol orcardiolipin (22). Titration of the calcium pump proteinwith phospholipids indicates that about 30 lipid mole-cules are required for full activation of the enzyme (23).
Magnetic resonance has been heavily exploited tostudy the problem of lipid binding to membrane pro-teins. The story has grown complex as the number ofstudies has multiplied. Many of the early studies usedelectron spin resonance (ESR) and spin-labeled lipids(24). The ESR spectra of these spin labels show twospectral components in membranes containing somemembrane proteins. Such data were interpreted interms of two lipid environments in the membrane inthe presence of membrane proteins. One spectral com-ponent was similar, though not identical, to the spectraobtained from spin labels in lipid bilayers without pro-tein. The other spectral component was representativeof a motion-restricted lipid environment. Model studiessuggested that the motion-restricted lipid environmentresulted from the lipid encountering the surface of pro-teins in the membrane. The population of the motion-restricted lipid environment was proportional to theprotein content of the membrane. Model calculationssuggested that the population of the motion-restrictedenvironment was suitable to form one ring of lipidaround the protein. (However, because recent studieshave indicated that many membrane proteins exist asdimers in the membrane (25), the details of these calcu-lations should be reexamined. For example, under con-ditions where the calcium pump protein is a dimer, thepopulation of the motion-restricted environment is ade-quate to surround the dimer with a single layer of lipid,but not enough to surround a monomer of the protein)(26). From these studies came the concept of a lipid an-nulus: lipid in a special environment produced by themembrane protein that is sufficient to cover the hydro-phobic surface of the protein.
After a period of these spin label studies, 2H-NMRstudies of 2H-labeled lipids in membranes containingproteins were used to examine the same questions. Thefirst such study suggested that results similar to those ofthe ESR studies would be obtained (27). However, thiswas later retracted in favor of results that revealed onlyone spectral component (28). The presence of one spec-tral component was interpreted variously in terms of1) no interactions between the lipids and the proteinsor 2) rapid exchange of lipids between sites next to theprotein and sites in the lipid bilayer of the membranegiving rise to a single, averaged spectral component.Considering the results and conclusions of the ESRstudies and the observations of lipids tightly bound tomembrane proteins, the first alternative is not a usefulinterpretation. The second interpretation rested on theabsence of a second spectral component in the 2H-NMRspectra. Subsequent publications demonstrated that acomponent resulting from lipids interacting stronglywith membrane proteins would not normally be visiblein the 2H-NMR spectra due to artifactual loss of sucha component in the collection of the data (29-31).Therefore, the interpretation of single-component 2H-NMR spectra in terms of lipid-protein interactions was
hazardous. Another approach or approaches to thequestion was required.
Phospholipid head groups contain the chemically in-teresting charged structures that might be expected tointeract with proteins (for example, choline of acetyl-choline that binds to the receptor of the same name, isalso found in the head group of one of the commonphospholipids), and so they are an important region ofthe lipid molecule to explore for a better understandingof lipid-protein interactions. 31P-NMR provides an ex-cellent, nonperturbing way to study the behavior of thelipid head groups.
31P-NMR has been used in the most recent studiesof lipid-protein interactions in biological membranesand reconstituted systems (32-34). In some cases, twospectral components are observed. One spectral com-ponent is similar, though not identical, to the spectrumarising from pure phospholipid bilayers. The otherspectral component is characteristic of a motion-restricted phospholipid head group environment.
These spectra can be deconvoluted into two spectralcomponents by subtraction of a normal bilayer compo-nent from the spectrum of the whole membrane. Theresulting difference spectrum is a broad (about twice asbroad as a normal phospholipid bilayer powder pat-tern) but axially symmetrical powder pattern that ischaracteristic of axial rotation, but indicative of a sub-stantially more ordered environment than is found inpure lipid bilayers. This latter spectral shape was ob-served most clearly from the phosphatidylinositoltightly bound to glycophorin reconstituted as a protein-phospholipid complex in a glycolipid bilayer (35). Inthis experiment, only the phospholipid that was tightlybound to the protein contributed to the 3tP-NMR spec-trum. So it was not necessary to use any of the assump-tions appropriate when spectra must be deconvolutedto obtain the individual components. However, decon-volution (that is, subtraction of the normal phospho-lipid bilayer powder pattern from the total spectrum)produced the same (as in the case of reconstituted glyco-phorin) broad axially symmetrical powder pattern ofprotein-bound phospholipid from the 31P-NMR spec-trum of the native and functional light sarcoplasmicreticulum membrane (B. S. Selinsky and P. L. Yeagle,unpublished results). Therefore, this broad (relative topure phospholipid bilayers) but axially symmetrical 3tPpowder pattern may be representative of the phospho-lipid head group environment when phospholipids arebound to membrane proteins in biological membranes.
Using magnetization transfer experiments, the ex-change rate of phospholipids between the motion-restricted environment and the lipid bilayer environ-ment was explicitly measured in sarcoplasmic reticu-lum in the only example to date of a direct measure-ment of such lipid exchange rates. An exchange rate of1 s1 was observed (36).
These more recent experiments appear to comple-ment the picture of lipid-protein interactions derivedfrom the ESR data. Both approaches identify a motion-restricted lipid environment induced by some mem-brane proteins. ESR data show that the environment is
1838 Vol. 3 May 1989 The FASEB Journal YEAGLE
enriched in particular phospholipids in the case ofNa,K-ATPase (37). 31P-NMR studies have revealed amodest exchange rate between the two lipid environ-ments for the sarcoplasmic reticulum, whereas forhuman erythrocyte glycophorin, the exchange rate isvery slow.
On the basis of these studies, the possibility of phos-pholipid regulation of membrane protein functionthrough binding to the membrane protein is still a via-ble hypothesis. However, the field has not progressedsufficiently to provide many complete examples of thishypothesis.
PROTEIN REGULATION OF
TRANSMEMBRANE LIPID DISTRIBUTION
One of the fascinating mysteries of membrane biologyis the creation and maintenance of transmembranelipid asymmetry. The erythrocyte membrane is thebest-documented example available of the asymmetri-cal distribution of phospholipid classes (38). The con-sensus of published data on the erythrocyte membraneis that the PE and phosphatidylserine (PS) are locatedon the inside (cytoplasmic face) of the membrane andthe sphingomyelin (SPM) is located almost entirely onthe exterior, whereas the PC is disproportionately dis-tributed toward the exterior face of the membrane.How these inhomogeneities of phospholipid distribu-tions are created or maintained had not previouslybeen explained. However, it has been known for sometime that transmembrane movement of lipids must oc-cur. The active sites of the enzymes involved in lipidbiosynthesis are found on the cytoplasmic face of theendoplasmic reticulum. Therefore, the newly synthe-sized lipid must be located initially on that cytoplasmicface of the membrane, and some select portion of thenewly synthesized pool must subsequently be translo-cated to the lumenal face of the endoplasmic reticulum.Several investigators have explored these important is-sues and shed new light on the problem of membranelipid asymmetry.
Recent experiments have revealed that proteinsprobably participate in the transmembrane movementof lipids and in the maintenance of the asymmetry oferythrocyte and endoplasmic reticulum membranes.Devaux (39) observed rapid movement in the erythro-cyte, outside to inside, of labeled phospholipid analogsof PE and PS. The movement is dependent on ATP.The PE and PS analogs compete with each other for thetransport system, with a greater affinity for the trans-locater shown by the PS analog. Sulfydryl group modi-fication inhibits the ability of the membrane to promotetranslocation of the PE and PS analogs. Results such asthese suggest that PE and PS translocation across theerythrocyte membrane is enhanced by a protein in themembrane. PC and sphingomyelin analogs translocatemuch more slowly by a pathway independent of thepathway for PE and PS translocation. Thus, the affinityof the transport system for these phospholipid analogsappears to be in the order: PS > PE >> PC, SPM. Such
a differential affinity is helpful in explaining the exclu-sive location of PS and the nearly exclusive location ofPE on the inside of the erythrocyte membrane. Theproteins involved in this translocation in the erythro-cyte have not been isolated, but preliminary efforts inthis area are under way.
A novel assay was developed to explore the samequestion regarding the endoplasmic reticulum (40).
Previous work showed evidence for relatively rapidtransmembrane movement of phospholipids in micro-somal preparations (41). More recently, the activity ofphospholipid translocation was reconstituted into lipidvesicles from rat liver microsomes. Although the pro-tein likely to be involved has not yet been identified, itappears that a new class of membrane proteins may beinvolved in facilitating the translocation of phospho-lipids across membranes. The question of which onesare energy dependent and which are not will have to beaddressed. From a thermodynamic view, translocationprocesses that are involved in the establishment and/ormaintenance of transmembrane lipid asymmetry pre-sumably require cellular energy.
LIPID REGULATION OF MEMBRANE
MORPHOLOGY
As indicated at the beginning of this review, it is pre-sumed that biological membranes have a lipid bilayeras their fundamental structural component. The lipidbilayer imparts the essential permeability control to thecell. Passive permeability through a lipid bilayer is slowfor most solutes (except water), so that membranetransport functions control the entry and exit ofnutrients from the cell. The loss of integrity of the lipidbilayer would make the cell freely permeable to allsolutes and lead to cell death.
The stability of the lipid bilayer is an important issueto explore in this context. It is well known that somelipids can readily adopt nonlamellar phase structures.Lipids that promote this behavior are detrimental tocell viability, although they probably have other impor-tant roles to play in cell membrane function (see thediscussion of stimulation of the Ca-ATPase by PE, ahexagonal (II) phase-forming lipid, above). In fact,aberrant lipid metabolism could lead to an imbalancebetween lamellar and nonlamellar phase-forming lipidsin a membrane and lead directly to a pathological stateinvolving membrane degeneration. It is important thento understand how lipids regulate the stability of a cellmembrane lipid bilayer.
The formation of hexagonal II phase by PE is a well-studied phenomenon. From its structure, one can read-ily conclude that the hexagonal II phase would be disas-trous to a cell membrane function. Three issues havebeen addressed recently concerning this phenomenonof nonlamellar phase formation: 1) the driving force forforming hexagonal II phase; 2) how a membrane mightadjust to maintain bilayer stability in the face of thepotential of nonlameilar phase formation; and 3) howmetabolic events within the cell might lead to at least
REGULATION OF CELL MEMBRANES 1839
transient bilayer instabilities that may have importantfavorable biological functions.
Forces governing hexagonal (II) phase formation
The formation of the hexagonal (II) phase structure hasbeen carefully studied by Gruner and colleagues (42)and by Siegel (43), who introduced the concept of in-trinsic radius of curvature, R0, to describe quantita-tively the formation of the cylinders of the hexagonal(II) phase. R0 reflects the radius of the channel formedby the close packing of the lipid head groups in the hex-agonal (II) phase. Thus, each phospholipid has an in-trinsic radius of curvature that can be measured byX-ray diffraction. Factors that affect the tendency of aphospholipid dispersion to enter the hexagonal (II)phase have predictable effects on R0.
A small value of R0 will favor the formation of thehexagonal (II) phase. For example, unsaturation in thelipid hydrocarbon chains favors the formation of thehexagonal (II) phase, and the hexagonal (II) phase isfavored at higher temperatures. Conversely, methyla-tion of the amino function will lead to an increase inR0 to the extent that R0 is nearly infinite for PC (i.e.,three methyl groups), which reflects the stability of thelamellar phase formed by PC.
This is part of the geometric argument that was ad-vanced previously, that PE is a wedge-shaped molecule(because of its small head group), and that wedge-shaped molecules pack well into the hexagonal (II)phase. However, new theories were called for when itwas reported that some lipids with large head groupsalso favored the hexagonal (II) phase (44). The latterresult suggested that the relative hydration of the headgroup, or its ability to interact with the aqueous phase,was an important physical property to explore.
A complementary thermodynamic viewpoint was de-veloped on the basis of related behavior of hexagonal(II) phase-forming lipids (20). It was observed that, inthe lamellar phase, membranes rich in PE tended to ag-gregate, membrane surface-to-membrane surface, topartially exclude water. This led to the conclusion thatthe surfaces of PE bilayers interacted poorly with theaqueous phase - a good example of the hydrophobiceffect. It could then be predicted that altering thenature of the aqueous phase should alter the extent ofsurface-surface interactions that were manifest as vesi-cle aggregation. Addition of chaotropic agents to theaqueous phase (which alter its nature) also interruptedthe aggregation of the PE vesicles. Furthermore, thechaotropic agents stabilized the lamellar phase of thePE against extremes in temperature. The tendency toform the hexagonal (II) phase can then be understoodas a thermodynamic consequence of the poor interac-tion of PE with the aqueous phase. Formation of thehexagonal (II) phase severely limits the exposure of thelipid head groups to water (they are exposed only to thesmall amount of water within the tubes and the headgroups are probably more tightly packed than on thesurface of the lamellar phase, again reducing their ex-
posure to water). Higher temperature or unsaturationincreases the surface area each head group is forced tooccupy in the lamellar phase, thereby exacerbating theunfavorable interaction of the PE bilayer surface withthe water. This fundamental physical property of PE,manifest as a modulation of bilayer surface properties,may be important to its role in biological membranesin the larnellar phase.
Biological response to hexagonal (II) phase potential
Acholeplasma laidlawii have MGDG and digalactosyldi-glyceride (DGDG) as major lipid components. Theselipids exhibit behavior analogous in some ways to PEand PC. MGDG is capable of forming the hexagonal(II) phase, as is PE. The head group of MGDG ispoorly hydrated, as is PE. In contrast, DGDG is analo-gous in some ways to PC: It is more extensivelyhydrated and is more stable in the lamellar phase thanis MGDG (45).
How do the Acholeplasma respond to stress induced byan increase in growth temperature? Increases in tem-perature will favor the hexagonal (II) phase of MGDG.Formation of hexagonal (II) phase would be disastrousto the organism. The response of the organism is todecrease the MGDG and increase the DGDG, or to de-crease the lipid favoring the hexagonal (II) phase andincrease the lipid favoring the lamellar phase. Structur-ally this alteration in composition will stabilize thelamellar phase of the lipids in the membrane againstthe temperature-induced tendency to form the hex-agonal (II) phase (46).
One of the big questions in this field concerns whatthe hexagonal (II) phase-forming lipid is doing in bio-logical membranes if one of its effects is to destabilizethe structure of those membranes. This is currently anarea of considerable interest and investigation.
Metabolic regulation of nonlamellar phase formation
A recent observation in the area of bilayer stability inbiological membranes is the ability of diacylglycerol toreduce dramatically the temperature of the lamellar-hexagonal (II) phase transition (47, 48). In some sys-tems, diacylglycerol levels as low as 1-3% (of the totallipid content) will reduce the lamellar-hexagonal (II)phase transition temperature by tens of degrees centri-grade. This reduction in the temperature of the lamellar-hexagonal (II) phase boundary can be understood interms of a reduction in R0. Therefore, diacylglycerol iscapable not only of activating protein kinase C, but alsoof disrupting the lipid bilayer of the membranes inwhich it is produced.
Diacylglycerol is an intermediate in lipid metabolismand can be produced by the action of phospholipase C.Phospholipase C can be stimulated by receptor activa-tion. The amount of diacylglycerol in the membranewill regulate the stability of the lipid bilayer of themembrane. Therefore, a mechanism for metabolicregulation of membrane stability through lipid metab-
1840 Vol. 3 May 1989 The FASEB Journal YEAGLE
olism is potentially available to the cell. As the discus-sion below suggests, such a pathway of regulation maybe important to biological membrane function.
Fusion of membranes and regulation by nonlamellar
phase formation
Cell membrane fusion is involved in a number of cellu-lar processes. For example, fusion is involved in receptor-mediated endocytosis and in intracellular vesiculartransport; it is involved in secretion and in viral infec-tion of some enveloped viruses.
At least three steps in the fusion process can be iden-tified: 1) the close approach of two membranes, oraggregation; 2) removal of at least part of the water be-tween the two membranes, or partial dehydration; and3) a transient destabilization of the lipid bilayer struc-ture to permit a partial, short-lived mixing of the struc-tures of the individual membranes that can lead tofusion of those membranes.
Lipid combinations that produce partial dehydrationof the membrane surface and are capable of introduc-ing transient nonlamellar structures into the lipidbilayer might be expected to enhance the incidence offusion. PE would be expected to be a good candidate inthis regard.
PE appears to enhance the rate of fusion in somemodel membrane systems. This may result in part fromthe facilitation by PE of the close apposition of the twomembranes to be fused.
However, an important new role that PE may play in
the fusion event has been identified (49). On compari-son of the phase behavior of a particular lipid system,it was suggested that fusion was greatly enhanced bythe so-called isotropic structures identified in the 31P-NMR spectra of that lipid system. These isotropicstructures occurred under the same conditions as thelipid particles in the freeze-fracture electron microscopyof the same preparations. The data suggested furtherthat if the system became hexagonal (II), fusion nolonger occurred and only extensive leakiness character-ized the membranes because of the breakdown of thelipid bilayer. So the isotropic lipidic particle appears tobe an important nonlamellar structure and may be acandidate for an intermediate in the fusion event.
In this context, the observations reviewed above-that diacylglycerol enhances the formation of the hex-agonal (II) phase and concurrently lowers the thresholdfor formation of the isotropic structures - suggest thatdiacylglycerol might enhance membrane fusion (47).Once again there is the possibility of a mechanism forthe regulation of membrane fusion that involves knownmetabolic events in the catabolism of cell membranelipids.
PROTEIN REGULATION OF MEMBRANE
MORPHOLOGY
It was noted above that lipids and proteins interact witheach other in biological membranes. One possible con-sequence of such interactions is that membrane pro-
teins might influence the morphological behavior ofmembrane lipids.
The retinal rod outer segment (ROS) disk mem-brane provides an interesting example of such aphenomenon. The membranes of the ROS disks arerich in PE and in highly unsaturated fatty acids. Infact, the single largest population of lipids in the diskmembrane is PE and the most abundant fatty acid is22:6. It was no surprise then to observe that the isolateddisk lipids were able to enter the hexagonal (II) phasein the presence of calcium (50). What was surprisingwas that the native membrane containing the photopig-ment rhodopsin was stable to more extreme stress (forexample, higher calcium concentration) than thatnecessary to produce hexagonal (II) phase in the iso-lated lipids. In this case the membrane proteins appearto stabilize the lipid bilayer of that membrane.
SUMMARY
In this review, we have examined some aspects of thelipid regulation of membrane function. The study ofcell membrane structure and function has progressedsufficiently to begin examining hypotheses for the regu-lation of membrane function through membrane struc-tural features. Through an examination of regulation,key issues of cellular biology begin to become un-raveled.
Studies of cholesterol regulation of Na,K-ATPase,as well as other membrane proteins, and of the regula-tion of cell growth have produced clues to the role ofcholesterol in mammalian cell biology. The availablecell biology and biochemistry studies suggest that cho-lesterol is required for the normal function of essentialmembrane enzymes. This is a structurally specific re-quirement. For example, in a cholesterol-requiring cell,ergosterol cannot substitute for cholesterol as the essen-tial sterol. The biochemical basis of this is the depen-dence of crucial membrane enzymes on the presence ofthe essential sterol in the membrane. Without the es-sential sterol, these crucial membrane enzymes cannotfunction, and without their function, the cell that re-quires those membranes cannot grow, divide, or differ-entiate. What remains to be described by future researchis the physical basis for the biochemical requirementcertain membrane enzymes have for cholesterol. Thestructural specificity of the sterol requirement suggeststhat the essential sterol binds to the enzyme that re-quires it at a sterol-specific binding site on the protein.The essential sterol then activates the enzyme as a posi-tive effector. However, this physical mechanism has yetto be established.
Understanding the role and mechanisms by whichmembrane phospholipids regulate membrane proteinactivity presents considerable challenges. Although theindividual species of lipids that are found in cellularmembranes number in the thousands, the biologicalreason for such variety has not been discovered. Andthe mechanism by which regulation of membrane pro-tein function (which has been observed for severallipids) occurs is still speculation. Much more work is re-
REGULATION OF CELL MEMBRANES 1841
quired. Factors to consider in such studies include notonly specific lipid-protein interactions but also what in-dividual lipids or combinations of lipids may do to thesurfaces of membranes and to membrane stability andinternal dynamics.
Studies of the regulation of lipid morphology andmechanisms of membrane fusion have led to new ideasabout the metabolic control of cellular membrane fu-sion. Intermediates discovered in the studies of phasechanges between lamellar and nonlamellar phases forlipids appear to facilitate membrane fusion. Otherstudies have shown that products (that can be generatedbiologically by receptor excitation) from lipid metabo-lism increase the incidence of the fusion-stimulatingstructural intermediates in the lipid membrane. Whatremains to be discovered is whether such a mechanismexists in vivo for biological control of membrane fusion.
Finally, research on the influence of phospholipidmetabolites on the stability of lipid bilayers suggests theexistence of regulatory mechanisms not only for nor-mal states of a cell or tissue, but also for disease states.If the balance between the lamellar phase and hexagonal(II) phase is upset, then membrane degeneration is theinevitable result. Cellular destruction would be thedirect consequence of such a disaster. L!1
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