Structure and Function of Sphingolipid- and Cholesterol-rich Membrane Rafts

melting temperature liquid crystalline liquid disordered liquid ordered phosphatidylethanolamine phosphatidylcholine detergent-resistant membrane glycosylphosphatidylinositol palmitoyl oleoyl PC T cell receptor It is well known that separate domains with different lipid compositions can exist in liposomes containing mixtures of different phospholipids. The question of whether cellular membranes contain similar lipid domains has intrigued workers for many years. One type of domain, sphingolipid and cholesterol-based structures called membrane rafts, has received much attention in the last few years. We will review the evidence that rafts exist in cells and focus on their structure, or the organization of raft lipids and proteins. Our discussion of function will focus on the role of rafts in signaling in hematopoietic cells, a particularly well developed area that has provided insights into raft organization in the membrane. Several reviews of rafts (1.Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8283) Google Scholar, 2.Brown D.A. London E. Annu. Rev. Cell Dev. Biol. 1998; 14: 111-136Crossref PubMed Scopus (2572) Google Scholar, 3.Brown D.A. London E. J. Membr. Biol. 1998; 164: 103-114Crossref PubMed Scopus (850) Google Scholar, 4.Jacobson K. Dietrich C. Trends Cell Biol. 1999; 9: 87-91Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar) and of related structures called caveolae (5.Anderson R.G.W. Annu. Rev. Biochem. 1998; 67: 199-225Crossref PubMed Scopus (1741) Google Scholar, 6.Kurzchalia T.V. Parton R.G. Curr. Opin. Cell Biol. 1999; 11: 424-431Crossref PubMed Scopus (516) Google Scholar, 7.Smart E.J. Graf G.A. McNiven M.A. Sessa W.C. Engelman J.A. Scherer P.E. Okamoto T. Lisanti M.P. Mol. Cell. Biol. 1999; 19: 7289-7304Crossref PubMed Scopus (929) Google Scholar) have appeared recently. Sphingolipids differ from most biological phospholipids in containing long, largely saturated acyl chains. This allows them to readily pack tightly together, a property that gives sphingolipids much higher melting temperatures (Tm)1 than membrane (glycero)phospholipids, which are rich in kinked unsaturated acyl chains. It is now clear that tight acyl chain packing is a key feature of raft lipid organization (3.Brown D.A. London E. J. Membr. Biol. 1998; 164: 103-114Crossref PubMed Scopus (850) Google Scholar, 8.Ahmed S.N. Brown D.A. London E. Biochemistry. 1997; 36: 10944-10953Crossref PubMed Scopus (621) Google Scholar, 9.Schroeder R.J. Ahmed S.N. Zhu Y. London E. Brown D.A. J. Biol. Chem. 1998; 273: 1150-1157Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar). In fact, the differential packing ability of sphingolipids and phospholipids probably leads to phase separation in the membrane. Thus, sphingolipid-rich rafts co-exist with phospholipid-rich domains that are in the familiar, loosely packed disordered state (variously abbreviated as Lα, lc, or ld). Phase separation between lipids in different physical states, most often the lc and the solid-like gel phases, has been well characterized in model membranes. Indeed, the gel phase is the most familiar state in which acyl chains are highly ordered. However, because of the high concentration of cholesterol in the plasma membrane and other membranes in which rafts form, raft lipids do not exist in the gel phase. Cholesterol has important effects on phase behavior. It is well known that addition of cholesterol to a pure phospholipid bilayer abolishes the normal sharp thermal transition between gel and lc phases, giving the membrane properties intermediate between the two phases. This effect initially suggested that domains in ordered and disordered states cannot co-exist at high cholesterol levels. However, further work showed that a different kind of phase separation can occur in binary mixtures of individual phospholipids with cholesterol. In these mixtures, domains in an lc-like phase co-exist with domains in a new state, the liquid-ordered (lo) phase. Acyl chains of lipids in the lo phase are extended and tightly packed, as in the gel phase, but have a high degree of lateral mobility (3.Brown D.A. London E. J. Membr. Biol. 1998; 164: 103-114Crossref PubMed Scopus (850) Google Scholar). Rafts probably exist in the lo phase or a state with similar properties. In support of this model, detergent-insoluble membranes that can be isolated from cell lysates and are likely to be derived from rafts (discussed below) are in the lo phase (10.Ge M. Field K.A. Aneja R. Holowka D. Baird B. Freed J. Biophys. J. 1999; 77: 925-933Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 11.Ostermeyer A.G. Beckrich B.T. Ivarson K.A. Grove K.E. Brown D.A. J. Biol. Chem. 1999; 274: 34459-34466Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Model membrane studies that do not involve detergents also support the idea that lo phase and lc phase domains could co-exist in biological membranes. These studies showed that phase separation can occur in ternary mixtures of cholesterol with two phospholipids (or a phospholipid and a sphingolipid) that have different Tm and thus different tendencies to form an ordered phase (8.Ahmed S.N. Brown D.A. London E. Biochemistry. 1997; 36: 10944-10953Crossref PubMed Scopus (621) Google Scholar, 12.Silvius J.R. del Guidice D. Lafleur M. Biochemistry. 1996; 35: 15198-15208Crossref PubMed Scopus (196) Google Scholar). In these mixtures, lo phase domains enriched in the high Tm lipid separate from lc phase domains enriched in the low Tm lipid. Because of the significant difference in Tm between sphingolipids and biological phospholipids, these lipid mixtures are a reasonable (though crude) model of cholesterol-containing cell membranes like the plasma membrane. Cholesterol has another important effect on phase behavior. As discussed above, there are parallels between lo/lc phase separation and gel/lcphase separation. In both cases, a phase in which acyl chains are highly ordered (gel or lo) separates from a phase in which they are disordered (lc). Thus, lipid mixtures can undergo either gel/lc phase separation in the absence of cholesterol or lo/lc phase separation in its presence. Comparing the phase behavior of mixtures with and without cholesterol shows that the sterol can sometimes promote phase separation (8.Ahmed S.N. Brown D.A. London E. Biochemistry. 1997; 36: 10944-10953Crossref PubMed Scopus (621) Google Scholar, 12.Silvius J.R. del Guidice D. Lafleur M. Biochemistry. 1996; 35: 15198-15208Crossref PubMed Scopus (196) Google Scholar), apparently because of favorable packing interactions between saturated lipids and sterol (13.Xu X. London E. Biochemistry. 2000; 39: 844-849Google Scholar). Thus, in phospholipid/sphingolipid mixtures, less sphingolipid is required to form the lo phase (in the presence of cholesterol) than to form the gel phase in its absence (8.Ahmed S.N. Brown D.A. London E. Biochemistry. 1997; 36: 10944-10953Crossref PubMed Scopus (621) Google Scholar, 9.Schroeder R.J. Ahmed S.N. Zhu Y. London E. Brown D.A. J. Biol. Chem. 1998; 273: 1150-1157Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar). This cholesterol effect probably explains why rafts can form in cell membranes that contain relatively low levels of sphingolipids. It also explains why cholesterol depletion can disrupt rafts and affect raft function. Finally, it probably explains why sphingomyelin, with aTm of 37–41 °C, can be essentially as effective as glycosphingolipids (which can have much higherTm) in promoting raft formation (11.Ostermeyer A.G. Beckrich B.T. Ivarson K.A. Grove K.E. Brown D.A. J. Biol. Chem. 1999; 274: 34459-34466Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Any difference in raft stability that might result from the difference inTm between the two lipids is minor compared with the strong raft-stabilizing effect of cholesterol. Because headgroup structure is an important modulator of lipid packing, headgroup as well as acyl chain structure may be important in raft formation. For instance, phosphatidylethanolamines (PE), with their small headgroup, have much higher Tm than the corresponding phosphatidylcholines (PC). This effect may be especially important in the sphingolipid-poor (but PE-rich) inner bilayer leaflet, where raft structure is very poorly understood. Membrane fragments that are insoluble in non-ionic detergents (DRMs; also termed DIGs (detergent-insoluble glycolipid-enriched membranes), GEMs (glycolipid-enriched membranes), and TIFF (Triton-insoluble floating fraction)) can be isolated from most mammalian cells (14.Brown D.A. Rose J.K. Cell. 1992; 68: 533-544Abstract Full Text PDF PubMed Scopus (2696) Google Scholar). DRMs appear to be derived from rafts; they are rich in cholesterol and sphingolipids and are in the lophase when isolated from cells (10.Ge M. Field K.A. Aneja R. Holowka D. Baird B. Freed J. Biophys. J. 1999; 77: 925-933Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Furthermore, lo phase liposomes are also detergent-insoluble under the conditions used to extract cells (9.Schroeder R.J. Ahmed S.N. Zhu Y. London E. Brown D.A. J. Biol. Chem. 1998; 273: 1150-1157Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar). Thus, there is a close relation between rafts and DRMs, and isolation of DRMs is one of the most widely used methods for studying rafts. The tight acyl chain packing of both gel and lo phase lipids is probably responsible for their detergent insolubility. This provides a rational explanation for the detergent insolubility of DRMs, which was initially puzzling; in a tightly packed state, lipid-lipid interactions can be more stable than lipid-detergent interactions. A number of proteins are enriched in DRMs. Some of these are targeted to rafts by modification with saturated chain lipid groups, which pack well into an ordered lipid environment. These modifications include glycosylphosphatidylinositol (GPI) anchors and closely spaced myristate and palmitate or dual palmitate chains (2.Brown D.A. London E. Annu. Rev. Cell Dev. Biol. 1998; 14: 111-136Crossref PubMed Scopus (2572) Google Scholar,15.Arni S. Keilbaugh S.A. Ostermeyer A.G. Brown D.A. J. Biol. Chem. 1998; 273: 28478-28485Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 16.Zhang W. Trible R.P. Samelson L.E. Immunity. 1998; 9: 239-246Abstract Full Text Full Text PDF PubMed Scopus (758) Google Scholar, 17.Melkonian K.A. Ostermeyer A.G. Chen J.Z. Roth M.G. Brown D.A. J. Biol. Chem. 1999; 274: 3910-3917Abstract Full Text Full Text PDF PubMed Scopus (558) Google Scholar, 18.Moffett S. Brown D.A. Linder M.E. J. Biol. Chem. 2000; 275: 2191-2198Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar). In contrast, both membrane-spanning proteins and prenyl groups (which are bulky and branched) should be difficult to accommodate in a highly ordered environment. Indeed, DRMs are relatively poor in transmembrane proteins and contain very low levels of prenylated proteins (17.Melkonian K.A. Ostermeyer A.G. Chen J.Z. Roth M.G. Brown D.A. J. Biol. Chem. 1999; 274: 3910-3917Abstract Full Text Full Text PDF PubMed Scopus (558) Google Scholar). Nevertheless, several specific transmembrane proteins are enriched in DRMs. Very little is known about how this occurs. Palmitoylation can contribute to DRM targeting (16.Zhang W. Trible R.P. Samelson L.E. Immunity. 1998; 9: 239-246Abstract Full Text Full Text PDF PubMed Scopus (758) Google Scholar, 17.Melkonian K.A. Ostermeyer A.G. Chen J.Z. Roth M.G. Brown D.A. J. Biol. Chem. 1999; 274: 3910-3917Abstract Full Text Full Text PDF PubMed Scopus (558) Google Scholar), although not all palmitoylated transmembrane proteins are in DRMs and not all transmembrane DRM proteins are palmitoylated. As might be expected, the sequence of the membrane-spanning domain (which could affect the way the protein interacts with lipids) can affect DRM localization (19.Perschl A. Lesley J. English N. Hyman R. Trowbridge I.S. J. Cell Sci. 1995; 108: 1033-1041Crossref PubMed Google Scholar, 20.Scheiffele P. Roth M.G. Simons K. EMBO J. 1997; 16: 5501-5508Crossref PubMed Scopus (572) Google Scholar, 21.Field K.A. Holowka D. Baird B. J. Biol. Chem. 1999; 274: 1753-1758Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). However, mutations in cytoplasmic domains, which seem unlikely to interact directly with lipids, can also affect DRM association (22.Puertollano R. Alonso M.A. J. Biol. Chem. 1998; 273: 12740-12745Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 23.Polyak M.J. Tailor S.H. Deans J.P. J. Immunol. 1998; 161: 3242-3248PubMed Google Scholar, 24.Brückner K. Labrador J. Scheiffele P. Herb A. Seeburg P. Klein R. Neuron. 1999; 22: 511-524Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar, 25.Machleidt T. Li W.-P. Liu P. Anderson R.G.W. J. Cell Biol. 2000; 148: 17-28Crossref PubMed Scopus (98) Google Scholar). Although the mechanism of this effect is not known, such mutants might fail to interact with binding partners that themselves associate directly with raft lipids or might be mistargeted to membranes whose lipid composition cannot support raft formation. The affinity of gangliosides (26.Hagmann J. Fishman P.H. Biochim. Biophys. Acta. 1982; 720: 181-187Crossref PubMed Scopus (47) Google Scholar) and lipid-linked proteins (15.Arni S. Keilbaugh S.A. Ostermeyer A.G. Brown D.A. J. Biol. Chem. 1998; 273: 28478-28485Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 27.Harder T. Scheiffele P. Verkade P. Simons K. J. Cell Biol. 1998; 141: 929-942Crossref PubMed Scopus (1054) Google Scholar) for DRMs (and presumably also for rafts) can be increased by clustering or oligomerization because of the increase in the number of saturated acyl chains per molecule or cluster. Enhancement of raft affinity by clustering of molecules that individually have more modest raft affinity is supported by theoretical considerations and may have important physiological consequences (2.Brown D.A. London E. Annu. Rev. Cell Dev. Biol. 1998; 14: 111-136Crossref PubMed Scopus (2572) Google Scholar,27.Harder T. Scheiffele P. Verkade P. Simons K. J. Cell Biol. 1998; 141: 929-942Crossref PubMed Scopus (1054) Google Scholar). This effect probably explains why several receptors on the surface of hematopoietic cells are recruited to DRMs when they are clustered following antigen binding (discussed below), although the structural features of these proteins that confer an affinity for rafts have not been identified. Although DRM association is a useful way of showing that a protein or lipid has an affinity for rafts, it cannot be used to quantitate the fraction of the molecule that is present in rafts in the intact cell. This is partly because cells must generally be chilled before detergent extraction in order to isolate DRMs. Chilling is necessary to stabilize the lophase and enhance its detergent resistance. However, because phase separation is also strongly temperature-dependent, more of the membrane is probably in the lo phase at 0 than at 37 °C. This may explain why a surprisingly high fraction of plasma membrane lipids can be detergent-insoluble (reviewed in Refs. 2.Brown D.A. London E. Annu. Rev. Cell Dev. Biol. 1998; 14: 111-136Crossref PubMed Scopus (2572) Google Scholar and 3.Brown D.A. London E. J. Membr. Biol. 1998; 164: 103-114Crossref PubMed Scopus (850) Google Scholar) and why the phospholipid composition of DRMs can be similar to that of the plasma membrane (28.Fridriksson E.K. Shipkova P.A. Sheets E.D. Holowka D. Baird B. McLafferty F.W. Biochemistry. 1999; 38: 8056-8063Crossref PubMed Scopus (248) Google Scholar). On the other hand, in some cases detergent may partially solubilize raft lipids and proteins even after chilling, leading to an underestimation of the fraction of these molecules in rafts (11.Ostermeyer A.G. Beckrich B.T. Ivarson K.A. Grove K.E. Brown D.A. J. Biol. Chem. 1999; 274: 34459-34466Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 15.Arni S. Keilbaugh S.A. Ostermeyer A.G. Brown D.A. J. Biol. Chem. 1998; 273: 28478-28485Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 29.Field K.A. Holowka D. Baird B. J. Biol. Chem. 1997; 272: 4276-4280Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). These temperature effects raise the question of whether rafts exist at all in cell membranes at physiological temperatures and highlight the importance of detergent-independent methods (described below) in providing evidence for the existence of rafts. If rafts are present in cells, it should be possible to detect them by microscopy. Nevertheless, molecules such as GPI-anchored proteins and gangliosides, taken as putative raft markers because of their enrichment in DRMs, often appear uniformly distributed on the cell surface when detected microscopically (reviewed in Refs. 3.Brown D.A. London E. J. Membr. Biol. 1998; 164: 103-114Crossref PubMed Scopus (850) Google Scholar and 4.Jacobson K. Dietrich C. Trends Cell Biol. 1999; 9: 87-91Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar). However, the distribution of these markers can change dramatically upon clustering with antibodies or other agents. In some cases, clustering of one marker can cause redistribution of another, although the two are unlikely to interact directly (2.Brown D.A. London E. Annu. Rev. Cell Dev. Biol. 1998; 14: 111-136Crossref PubMed Scopus (2572) Google Scholar, 27.Harder T. Scheiffele P. Verkade P. Simons K. J. Cell Biol. 1998; 141: 929-942Crossref PubMed Scopus (1054) Google Scholar, 30.Viola A. Schroeder S. Sakakibara Y. Lanzavecchia A. Science. 1999; 283: 680-682Crossref PubMed Scopus (844) Google Scholar, 31.Janes P.W. Ley S.C. Magee A.I. J. Cell Biol. 1999; 147: 447-461Crossref PubMed Scopus (703) Google Scholar, 32.Schütz G.J. Kada G. Pastushenko V.P. Schindler H. EMBO J. 2000; 19: 892-901Crossref PubMed Scopus (490) Google Scholar) (discussed below in the section on hematopoietic cell signaling). The implication that the two markers colocalize because both are associated with rafts provides some of the strongest evidence to date that rafts are present in cell membranes. Why are rafts difficult to see without clustering? At least three explanations are possible. First, rafts may be too small to see by microscopy. If so, clustering might cause coalescence of rafts into larger, visible units. Second, if individual markers have only a moderate affinity for rafts, they may not be highly concentrated in the domains. For instance, rafts would be difficult to detect visually if the concentration of a marker there were only 3- or 4-fold higher than in the rest of the membrane. These values are plausible because phase diagrams indicate that saturated acyl chains can sometimes give lipids only a moderate preference for ordered membrane domains (33.Mabrey S. Sturtevant J.M. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 3862-3866Crossref PubMed Scopus (965) Google Scholar). As discussed earlier, clustering could increase the affinity of these markers for rafts, increasing their concentration in the domains and facilitating detection. Finally, it is even possible that rafts do not exist constitutively but that clustering of components that have an affinity for an ordered state induces raft formation. We have discussed these alternate models of raft structure and dynamics previously (3.Brown D.A. London E. J. Membr. Biol. 1998; 164: 103-114Crossref PubMed Scopus (850) Google Scholar), and they are illustrated in Fig. 1. As is clear from the previous discussion, raft size is very poorly understood. Microscopically detectable lipid domains can sometimes be observed in model membranes without cholesterol (34.Korlach J. Schwille P. Webb W.W. Feigenson G.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8461-8466Crossref PubMed Scopus (731) Google Scholar, 35.Bagatolli L.A. Gratton E. Biophys. J. 2000; 78: 290-305Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar), but it is not yet clear how cholesterol affects domain size. Single-particle tracking experiments in cells showed that a ganglioside and a GPI-anchored protein were transiently confined to domains of about 200–300 nm that were sensitive to a glycosphingolipid synthesis inhibitor (4.Jacobson K. Dietrich C. Trends Cell Biol. 1999; 9: 87-91Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar). Two other groups have used resonance energy transfer to search for clustering of GPI-anchored proteins in the membrane (36.Varma R. Mayor S. Nature. 1998; 394: 798-802Crossref PubMed Scopus (1045) Google Scholar,37.Kenworthy A.K. Petranova N. Edidin M. Mol. Biol. Cell. 2000; 11: 1645-1655Crossref PubMed Scopus (394) Google Scholar). One group (36.Varma R. Mayor S. Nature. 1998; 394: 798-802Crossref PubMed Scopus (1045) Google Scholar) found cholesterol-dependent clustering into domains of <70 nm, too small to see by microscopy, whereas the other found no evidence for clustering (37.Kenworthy A.K. Petranova N. Edidin M. Mol. Biol. Cell. 2000; 11: 1645-1655Crossref PubMed Scopus (394) Google Scholar). This discrepancy would be reconciled if in fact only a small fraction of the molecules were closely clustered, a possibility that is consistent with both studies (37.Kenworthy A.K. Petranova N. Edidin M. Mol. Biol. Cell. 2000; 11: 1645-1655Crossref PubMed Scopus (394) Google Scholar). It should be noted, however, that preferential partitioning of proteins into rafts does not necessarily result in their close clustering. For instance, GPI-anchored proteins could be present at a relatively low concentration in rafts if they had only a moderate affinity for the domains. Furthermore, as there is no reason to suspect that GPI-anchored proteins in rafts interact with each other more strongly than with raft lipids, they may be uniformly distributed within rafts. Thus, GPI-anchored proteins could be present at low local density if a large fraction of the membrane formed rafts. Rafts are likely to be most abundant in membranes that are rich in cholesterol and sphingolipids, including the plasma membrane, late secretory pathway, and endocytic compartments. In the plasma membrane, rafts appear to have a preferential association with 50–100-nm pits called caveolae, which are present in many mammalian cell types (5.Anderson R.G.W. Annu. Rev. Biochem. 1998; 67: 199-225Crossref PubMed Scopus (1741) Google Scholar, 6.Kurzchalia T.V. Parton R.G. Curr. Opin. Cell Biol. 1999; 11: 424-431Crossref PubMed Scopus (516) Google Scholar, 7.Smart E.J. Graf G.A. McNiven M.A. Sessa W.C. Engelman J.A. Scherer P.E. Okamoto T. Lisanti M.P. Mol. Cell. Biol. 1999; 19: 7289-7304Crossref PubMed Scopus (929) Google Scholar). It should be noted, though, that rafts (as detected by DRM formation and by the co-clustering of several putative raft components) are not restricted to caveolae and are abundant in cells that lack caveolae. It is not yet known whether localization in caveolae affects the structure of rafts. The behavior of rafts in the cytoplasmic leaflet of the bilayer is an important outstanding question. Although sphingolipids are largely restricted to the outer leaflet, several observations suggest that rafts are present in the inner leaflet and that rafts in the two leaflets are coupled. First, DRMs have a bilayer appearance when isolated and are enriched in dually acylated cytofacial membrane proteins, such as Src family kinases and G protein α subunits. Second, Src family kinases can co-redistribute when cell-surface GPI-anchored proteins or gangliosides are clustered using antibodies or toxins (27.Harder T. Scheiffele P. Verkade P. Simons K. J. Cell Biol. 1998; 141: 929-942Crossref PubMed Scopus (1054) Google Scholar, 31.Janes P.W. Ley S.C. Magee A.I. J. Cell Biol. 1999; 147: 447-461Crossref PubMed Scopus (703) Google Scholar). Recent advances in tandem electrospray mass spectrometry have shown that plasma membrane phospholipids are more highly saturated than those in intracellular membranes (28.Fridriksson E.K. Shipkova P.A. Sheets E.D. Holowka D. Baird B. McLafferty F.W. Biochemistry. 1999; 38: 8056-8063Crossref PubMed Scopus (248) Google Scholar, 38.Schneiter R. Brügger B. Sandhoff R. Zellnig G. Leber A. Lampl M. Athenstaedt K. Hrastnik C. Eder S. Daum G. Paltauf F. Wieland F.T. Kohlwein S.D. J. Cell Biol. 1999; 146: 741-754Crossref PubMed Scopus (389) Google Scholar) and thus may form rafts readily in the presence of cholesterol. The monounsaturated phospholipid palmitoyl oleoyl phosphatidylcholine (POPC) may form the lo phase when mixed with cholesterol (39.Mateo C.R. Acuña A.U. Brochon J.-C. Biophys. J. 1995; 68: 978-987Abstract Full Text PDF PubMed Scopus (132) Google Scholar). In addition, mixtures of brain PC (which is rich in POPC) and cholesterol are partially Triton-insoluble (in the cold) in the absence of sphingolipids (9.Schroeder R.J. Ahmed S.N. Zhu Y. London E. Brown D.A. J. Biol. Chem. 1998; 273: 1150-1157Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar). Finally, as mentioned earlier, the high concentration of relatively highTm PE in the inner leaflet may promote raft formation. Together, these observations suggest that formation of lo phase rafts in the sphingolipid-poor cytoplasmic membrane leaflet is plausible. How these rafts might be coupled with outer leaflet rafts is very poorly understood. There is some evidence for monolayer coupling of sphingolipid-rich domains in model membranes (40.Schmidt C.F. Barenholz Y. Huang C. Nature. PubMed Scopus Google Scholar). and were used to a between like in the two leaflets of model membranes containing two phospholipid (34.Korlach J. Schwille P. Webb W.W. Feigenson G.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8461-8466Crossref PubMed Scopus (731) Google Scholar). of the of this effect will probably on the behavior of rafts in