Jumping to rafts: gatekeeper role of bilayer elasticity

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1 Review TRENDS in Biochemical Sciences Vol.29 No.6 June 2004 Jumping to rafts: gatekeeper role of bilayer elasticity Daniel Allende 1,2, Adriana Vidal 1 and Thomas J. McIntosh 1 1 Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA 2 Department of Neurobiology, Duke University Medical Center, Durham, NC 27710, USA Two of the physiologically important processes that take place in biological membranes are the partitioning of water-soluble proteins into the membrane and the sequestering of specific transmembrane proteins into membrane microdomains or rafts. Although these two processes often involve different classes of protein, recent biophysical studies indicate that they both strongly depend on the structural and elastic properties of the membrane bilayer. That is, both the partitioning of peptides into membranes and the distribution of transmembrane peptides in the plane of the membrane are modulated by physical properties of the lipid bilayer that are controlled by cholesterol content and the composition of the phospholipid hydrocarbon chain. Biological membranes contain many types of lipid, including several classes of phospholipid and glycolipid, as well as variable concentrations of cholesterol. Lipid content varies according to the specific membranous organelle; for example, sphingolipids and cholesterol are enriched in plasma membranes but nearly absent in endoplasmic reticulum membranes. Moreover, membrane lipids differ widely in hydrocarbon chain composition, with variations in hydrocarbon chain length and the number of double bonds per chain. For cell biologists and membrane biochemists, a longstanding conundrum concerns the reasons why membranes contain so many types of lipid [1,2]. In terms of protein lipid interactions and many biological functions, it is now clear that two distinct features of membrane lipids are important: chemically specific properties of the individual lipid molecule, in particular those of the headgroup; and structural and elastic (material) properties of the whole bilayer (or microdomains in the bilayer), which are primarily determined by the composition of the lipid hydrocarbon chain. In biological processes, specific types of lipid molecule have different roles [2]. Inositol and choline phospholipids are sources of second messengers in transmembrane signaling [3,4]; phosphatidylcholine provides optimal functioning of 3-hydroxybutyrate dehydrogenase [5]; cardiolipin supports mitochondrial inner membrane integrity and proton conduction [6]; phosphatidylethanolamine and phosphatidylglycerol enhance the activity of a bacterial signal peptidase [7]; negatively charged phospholipids bind peptides and proteins to the membrane Corresponding author: Thomas J. McIntosh (T.McIntosh@cellbio.duke.edu). Available online 6 May 2004 surface [8]; and glycolipids interact with lectins and toxins, and are involved in cell cell contacts [9]. In addition, many studies have shown that the composition of the hydrocarbon chain region of the bilayer is important in lipid protein interactions and in modulating membrane functions. For example, the activities of specific membrane pumps and transporters incorporated into bilayers depend on the length of the phospholipid acyl chain [10,11]; and protein binding to membranes [12] and the activity of enzymes and important membrane proteins, such as rhodopsin [13,14], are modulated by unsaturation in the phospholipid hydrocarbon chain. Moreover, specific membrane proteins are sequestered into specialized regions of plasma and Golgi membranes, called microdomains or rafts, which have a more ordered hydrocarbon interior than the surrounding bilayer [15 17]. This sequestration of some proteins into rafts, coupled with the exclusion of others [18], is thought to be essential to several membrane functions, including signal transduction [18 21], membrane fusion [22] and protein trafficking [15,23]. Here we discuss recent studies on the role of the structural and mechanical properties of bilayers in two aspects of peptide lipid interactions: first, the partitioning of physiologically important peptides from the aqueous phase into membranes; and second, the distribution of transbilayer peptides in the plane of the bilayer and the mechanisms involved in sorting peptides into or out of membrane rafts. Our particular focus is the role of cholesterol in these peptide lipid interactions, because cholesterol modifies both the structural and the elastic properties of bilayers. In short, cholesterol decreases the area per phospholipid molecule [24], increases bilayer hydrophobic thickness [24] and increases the bilayer area compressibility modulus (K a ), a measure of the cohesion between lipid molecules obtained from stress versus strain plots [25]. Partitioning of water-soluble peptides into bilayers Several diverse classes of water-soluble protein and peptide bind to both biological membranes and lipid bilayers [26 29]. Of such peptides, one of the best characterized is melittin, a cationic peptide of 26 amino acids from honeybee venom that induces channel formation in bilayers [30]. The interactions between melittin and bilayer vesicles have been analyzed by several techniques. Particularly useful for comparing bilayer systems are measurements of the partition coefficient and the resulting free energy of transfer (DG o ) of melittin /$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi: /j.tibs

2 326 Review TRENDS in Biochemical Sciences Vol.29 No.6 June 2004 Table 1. Free energy of melittin transfer and melittin-induced leakage for large unilamellar vesicles a,b Lipid DG o (kcal/mol) Percentage of leakage EPC 27.6 ^ ^ 1.9 EPC:cholesterol (1:1) 26.4 ^ ^ 0.9 Sphingomyelin:cholesterol (1:1) 24.5 ^ ^ 1.0 EPC: phosphatidylserine (85:15) 28.9 ^ ^ 2.0 LPS ^ ^ 0.5 a Abbreviations: DG o, free energy of transfer; EPC, egg phosphatidylcholine; LPS, lipopolysaccharide. b Data are taken from Ref. [34]. from water to bilayer [31], and the melittin-induced leakage of small water-soluble fluorescent probes encapsulated in the vesicle interior [32,33]. Values of DG o provide a quantitative measurement of the strength of binding (the more negative the DG o value, the larger the binding), whereas leakage data provide a functional assay for this membrane-lytic peptide. Several studies have characterized melittin binding and melittin-induced leakage for vesicles composed of lipids that are typical of eukaryotic membranes. For example, for electrically neutral bilayers comprising palmitoyloleoylphosphatidylcholine (POPC) or egg phosphatidylcholine (EPC), DG o is between 27 and 28 kcal/mol [31,34]. As shown in Table 1, the addition of negatively charged lipids, such as phosphatidylserine, increases melittin partitioning through electrostatic attraction [31,34], whereas the addition of cholesterol decreases partitioning [34,35]. The melittin-induced leakage is not simply related to the measured partitioning of melittin, however, because the addition of either negatively charged lipids [32 34] or cholesterol [34,35] reduces melittin-induced vesicle leakage (Table 1). For the negatively charged lipids, a possible explanation for the low leakage is that electrostatic attraction between the positively charged melittin and the negatively charged lipids keeps the peptide at the bilayer interface, thereby preventing the formation of a transbilayer peptide pore [32,33]. For cholesterol, the low binding and leakage probably occur because cholesterol tightens the packing of hydrocarbon chains in the bilayer (i.e. reduces the area per molecule and the area per lipid hydrocarbon chain) and increases the area compressibility modulus (K a ) [36]. The area per molecule and K a might be expected to have an important role in peptide partitioning into the bilayer because, for an amphipathic peptide to maximize hydrophobic interactions with a bilayer, it would have to partition into the interfacial region where it would occupy space. There is less initial volume available in the interfacial region when the area per molecule is reduced, and K a is a measure of the energy needed to dilate the bilayer surface area. A further demonstration of the role of area compressibility is the extremely low binding of melittin to bilayers composed of equimolar sphingomyelin and cholesterol (Table 1): such bilayers have much larger values of K a ( dyn/cm) than have other phospholipid or phospholipid:cholesterol bilayers [25]. As shown in Figure 1, a quantitative relationship between peptide binding and K a G o (kcal/mol) Melittin MPR K a (dyn/cm) Figure 1. Plot of the free energy of transfer (DG o ) for melittin and the presequence of mitochondrial protein rhodanese (MPR) into lipid vesicles as a function of the magnitude of the bilayer area compressibility modulus (K a ). The experimental points (in order of increasing values of K a ) are egg phosphatidylcholine (EPC), 1:1 EPC:6-ketocholestanol, 1:1 EPC:cholesterol, and 1:1 sphingomyelin:cholesterol. The binding of MPR to 1:1 sphingomyelin:cholesterol is too small to determine DG o accurately [36]. The unbroken and broken lines are the least-squares fits to the melittin and MPR data, respectively. Data are taken from Ref. [36]. is obtained for melittin and for another water-soluble amphipathic peptide, the presequence of mitochondrial protein rhodanese (MPR). For both peptides, the magnitude of DG o decreases linearly with increasing K a [36]. These binding and leakage experiments have been recently extended to lipids found on the surface of bacterial membranes. Besides their cytoplasmic membrane, Gramnegative bacteria such as Escherichia coli contain an outer membrane, which provides an additional barrier in bacterial antibiotic resistance and protects strains of these bacteria against melittin and several antimicrobial peptides [37]. This outer membrane has an unusual lipid composition in that its outer monolayer contains high concentrations of lipopolysaccharide (LPS), a complex molecule containing polysaccharides covalently linked to a lipid moiety (lipid A), which usually contains six or seven hydrocarbon chains. The magnitude of DG o is relatively large for LPS bilayers (Table 1) owing to electrostatic interactions between melittin and the negative charges on LPS [34]. ForLPSbilayers,however,themelittininduced leakage is small, and is similar to that observed for 1:1 EPC:cholesterol bilayers. Allende and McIntosh [34] argue that the tight hydrocarbon chain packing (small area per hydrocarbon chain) measured for LPS bilayers [38], which is similar to the tight chain packing in EPC:cholesterol bilayers [24], explains the resistance of LPS to melittin-induced leakage. Thus, owing to tight hydrocarbon chain packing, both cholesterol in eukaryotic plasma membranes and LPS in bacterial outer membranes provide protection against the lytic effect of melittin [34].

3 Review TRENDS in Biochemical Sciences Vol.29 No.6 June G def (kcal/mol) Number of residues Figure 2. Theoretical calculation of the free energy of deformation (DG def ) for the insertion of a-helices into bilayers. The DG def is shown for a-helices with transmembrane domains (TMDs) of amino acid residues inserting into stearoyloleoylphosphatidylcholine (SOPC) bilayers (filled circles), 1:1 SOPC:cholesterol bilayers (filled squares), and a bilayer with a thickness corresponding to 1:1 SOPC: cholesterol but with the material properties of SOPC (open squares). Redrawn, with permission, from Ref. [63]. Sequestration of transmembrane peptides into detergent-resistant rafts For both biological membranes and bilayers containing lipid compositions similar to those found in cell plasma membranes [39,40], microdomains or rafts have been characterized by their insolubility at low temperatures to detergents such as Triton X-100 [21,39,41,42]. It has been found that these detergent-resistant membranes (DRMs) are enriched in specific lipids including sphingomyelin and cholesterol [15,16,42 44], whereas non-raft or detergentsoluble membranes (DSMs) are enriched in phospholipids containing more double bonds per molecule as compared with sphingomyelin [42,45]. Bilayers composed of sphingomyelin and cholesterol have different structural and mechanical properties from those of bilayers comprising unsaturated phospholipids typically found in membranes. For example, recent X-ray diffraction studies [42] have shown for a 1:1:1 mixture of dioleoylphosphatidylcholine (DOPC):sphingomyelin: cholesterol that the hydrocarbon thickness of DRMs (enriched in sphingomyelin:cholesterol) is 9 Å (,30%) greater than that of DSMs (enriched in DOPC, which has one double bond per hydrocarbon chain). In addition, as noted above, micropipette aspiration experiments have shown that the K a of equimolar sphingomyelin:cholesterol bilayers is more than 1700 dyn/cm [25] or about nine times the value of K a measured for DOPC bilayers [46]. Role of bilayer thickness in protein lipid interactions The thickness of raft and non-raft bilayers might have an important role in the sorting of transmembrane proteins because of the effects of hydrophobic mismatch between the thickness of the bilayer hydrocarbon region and the length of the transmembrane domain (TMD) of the protein, as described in the classical mattress model of Mouritsen and Bloom [47]. In other words, if there were a mismatch between the length of the TMD and the hydrocarbon thickness, then the bilayer would have to deform to prevent the exposure of hydrophobic amino acid residues or lipid methylene groups to water [47]. As detailed below, such a deformation would be energetically unfavorable. The difference in thickness of raft and non-raft membranes has been proposed to be a factor in membrane protein trafficking through the Golgi apparatus, where rafts are first formed [17]. It has been suggested that in the Golgi apparatus resident Golgi proteins with relatively short TMDs are localized in thin non-raft membranes, whereas proteins with longer TMDs are segregated to thicker microdomains that are enriched in sphingomyelin and cholesterol and that form transport vesicles destined for the plasma membrane [48,49]. Although other factors, such as the specific amino acid composition of the proteins, seem to be involved in protein sorting in the Golgi [50], the importance of TMD length in protein trafficking is indicated by experiments showing that the cell localization of specific membrane proteins depends on their TMD length [49,51,52]. Consistent with a physiological role for the hydrophobic matching of TMD length and bilayer thickness are many recent experiments with lipid membranes showing that the amount of transmembrane peptide incorporated into bilayers and the orientation of the peptides in the bilayer depend on the extent of the hydrophobic mismatch [53 56]. In addition, the functioning of gramicidin channels has been shown to depend on bilayer thickness [57 59]. Role of cholesterol in hydrophobic mismatch and bilayer deformation The energetic cost of bilayer deformation (DG def ) caused by hydrophobic mismatch between TMD length and hydrocarbon thickness should crucially depend on the elastic properties of the bilayer, in particular K a and the related bilayer bending modulus (B) [60,61]. That is, for a bilayer to deform locally to match the length of a protein s TMD, the area per molecule of the lipids around the protein must change and the bilayer must bend locally [60 62]. Experimental values of B and K a have now been determined for various bilayer systems [25,46]. A recent detailed theoretical treatment has quantitatively predicted DG def values and shown that the sorting of proteins with different TMD lengths between raft and nonraft membranes depends on both the relative hydrocarbon thicknesses and the material properties (K a and B) of raft and non-raft bilayers [63]. Figure 2 shows theoretical predictions of DG def for the insertion of a-helical peptides of different hydrophobic lengths into bilayers composed of stearoyloleoylphosphatidylcholine (SOPC) and equimolar SOPC:cholesterol. For SOPC, DG def decreases with an increasing number of amino acids in the helix, because hydrophobic matching occurs for peptides containing a TMD of about 20 amino acids. The addition of cholesterol increases the magnitude of DG def for peptide lengths that

4 328 Review TRENDS in Biochemical Sciences Vol.29 No.6 June 2004 (a) DSM (b) DRM 0.15 P-23 P-29 P Å P Å Figure 3. Illustration of peptides with different lengths in detergent-soluble membranes (DSMs) and detergent-resistant membranes (DRMs). Peptides P-23 [KKG(LA) 4 W(LA) 4 KKA] and P-29 [KKG(LA) 5 LW(LA) 5 LKKA] are shown in bilayers with widths corresponding to DSMs (a) and DRMs (b), respectively, that were isolated from 1:1:1 dioleoylphosphatidylcholine (DOPC):sphingomyelin:cholesterol bilayers [65]. The peptides were designed so that the transbilayer region (17 and 23 non-polar amino acid residues for P-23 and P-29, respectively) matched the hydrophobic thicknesses of DSMs and DRMs, respectively, obtained from X-ray diffraction analysis [42]. Phospholipids are shown as circular headgroups (dark blue, sphingomyelin; light blue, DOPC) attached to wavy hydrocarbon chains; cholesterol is shown as an open oval; and the peptides are shown as rectangles. In each peptide, the open central box corresponds to the transbilayer a-helical core region and the red boxes correspond to the hydrophilic regions, which each contain two lysine residues that anchor the hydrophilic ends of the peptide to the interfacial region. Similar peptides were used in Ref. [64], except that each hydrophilic anchoring region contained two tryptophan residues and the transbilayer segments contained 17, 21 or 25 amino acid residues. show hydrophobic mismatch, primarily because cholesterol increases the values of K a and B [63]. In an elegant demonstration of this point, Lundbaek et al. [63] made calculations for SOPC:cholesterol bilayers showing that if cholesterol increases bilayer thickness without changing K a and B (Figure 2, open squares), there would be much smaller changes in DG def. Their analysis further predicts that the distribution of proteins between cholesterol-enriched and cholesterol-depleted microdomains can be regulated by these cholesterol-induced changes in the material properties of the bilayers. The energy calculations of Lundbaek et al. [63] indicate that cholesterol-induced changes in bilayer properties can be an effective mechanism for sorting proteins between raft and non-raft bilayers and that cholesterol-induced protein sorting can be a proofreading mechanism that excludes short TMDs from entering rafts, consistent with the idea that proteins with short TMDs are retained in the Golgi apparatus [48]. Experimental studies of peptide sorting by TMD length Recent studies have examined whether peptides with different TMD lengths can indeed be sorted into or out of rafts [64,65]. These experiments determined the distribution of transmembrane peptides designed to have TMDs with lengths matching the hydrocarbon thicknesses of either the DSMs or the DRMs isolated from DOPC: sphingomyelin:cholesterol bilayers [42]. The peptides and lipid bilayers used in these studies are shown in Figure 3. In one set of experiments, DSMs and DRMs were isolated by Triton X-100 treatment at 4 8C, and the presence of the peptides in DSMs and DRMs was determined by thin-layer chromatography [64]. Peptides of different lengths, either with or without palmitate chains covalently linked to their amino (N)-termini, are Peptide:lipid (mol ratio) DSM DRM DSM DRM 4 o C 37 o C Figure 4. Ratio of peptide to total lipid in detergent-soluble membranes (DSMs) and detergent-resistant membranes (DRMs) for both P-23 and P-29 at 4 8C and 37 8C. Data are taken from Ref. [65]. found to be located primarily in DSMs, indicating that neither hydrophobic matching nor peptide palmitoylation is sufficient to promote the partitioning of transmembrane peptides into DRMs [64]. This non-effect of transmembrane peptide palmitoylation is interesting, because palmitoylation has been shown to target small peptides [66] or cytoplasmic G proteins [20,67] to rafts. van Duyl et al. [64] argue that the relatively tight hydrocarbon packing of liquid-ordered DRMs provides an unfavorable environment for accommodating protein TMDs. The above experiments have been extended by the use of chemical assays to determine quantitatively the molar concentrations of peptide and types of lipid in DSMs and DRMs isolated at different temperatures [65]. At48C (the temperature at which most detergent solubility experiments are done with cells) and 37 8C (physiological temperature), the distributions of lipid are found to be similar. At both temperatures, the DRMs are enriched in sphingomyelin and cholesterol, and the DSMs are enriched in DOPC [65]. At both temperatures, the short (P-23) and long (P-29) peptides are preferentially localized to the DSMs (Figure 4). This result demonstrates the importance of the mechanical properties of the bilayer relative to hydrophobic mismatch. At 37 8C, however, there is considerably more P-29 than P-23 in the DRMs (Figure 4), indicating that detergent solubility experiments done at 4 8C might not give a completely accurate view of the concentration of specific protein or peptides in rafts and also that hydrophobic matching has a role in peptide sorting at physiological temperatures. A key factor for these observations might be the temperature dependence of the structural and material properties of raft and non-raft bilayers [65]. Consistent with this idea are recent microscopy [40,68,69] and NMR [70,71] studies showing that the morphology, formation and lipid lateral diffusion parameters of bilayer microdomains are dependent on temperature.

5 Review TRENDS in Biochemical Sciences Vol.29 No.6 June Table 2. Mole-fraction partition coefficients and apparent free energies of transfer for peptides and lipids from DSMs to DRMs at 37 8C a,b Molecule K p DG a (kcal/mol) P ^ ^ 0.13 P ^ ^ 0.16 Cholesterol 1.58 ^ ^ 0.15 DOPC 0.15 ^ ^ 0.39 Sphingomyelin 4.26 ^ ^ 0.20 a Abbreviations: DG a, apparent free energy of transfer; DOPC, dioleoylphosphatidylcholine; K p, partition coefficient; P-23, transbilayer peptide with 23 amino acids; P-29, transbilayer peptide with 29 amino acids. b Data are taken from Ref. [65]. In the same study [65], the apparent free energies of transfer (DG a ) of peptides and lipid components from DSMs to DRMs were calculated from the measured molar concentrations of the lipids and peptides in the DSMs and DRMs. (The energies were denoted apparent because the calculations made the assumption that detergent isolation does not alter the distribution of the lipids or peptides. A positive value of DG a means that the transfer of the molecule from a DSM to a DRM is energetically unfavorable.) As shown in Table 2, at physiological temperature the absolute values of DG a for cholesterol and P-29 are found to be less than the magnitude of thermal energy (0.6 kcal/mol). In this regard, recent NMR data [70] point to the rapid exchange of cholesterol between different membrane regions in phosphatidylcholine:sphingomyelin:cholesterol bilayers, although other NMR studies indicate that the exchange of lipids between domains seems to be slow on the timescale of lateral diffusion measurements because of the large area of the domains [71]. The data in Table 2 imply that raft formation represents only a marginally effective mechanism for membrane sorting of cholesterol and P-29. By contrast, at physiological temperature the magnitude of DG a is considerably larger than thermal energy for DOPC, sphingomyelin and P-23 (Table 2), indicating that raft formation may have a significant impact on the sorting of unsaturated phosphatidylcholine, sphingomyelin and proteins with relatively short TMDs [65]. Concluding remarks Recent experiments and theoretical treatments indicate that bilayer structure and elastic properties have important roles both in modulating the partitioning of watersoluble peptides into bilayers and in the localization of transmembrane peptides in bilayer microdomains. The relevant structural and elastic properties depend on specific features of bilayer lipid composition, particularly the presence of cholesterol, sphingomyelin or phospholipids with multiple double bonds. Acknowledgements We thank Sidney Simon for many stimulating discussions and helpful comments. This work was supported by NIH grants GM27278 and GM References 1 Dowhan, W. 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