Jumping to rafts: gatekeeper role of bilayer elasticityPartitioning of water-soluble peptides into bilayersSequestration of transmembrane peptides into detergent-resistant raftsRole of bilayer thickness in protein-lipid interactionsRole of cholesterol in hydrophobic mismatch and bilayer deformationExperimental studies of peptide sorting by TMD lengthConcluding remarksAcknowledgementsReferencesJumping to rafts:gatekeeper role of bilayer elasticityDaniel Allende1,2, Adriana Vidal1and Thomas J. McIntosh11Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA2Department of Neurobiology, Duke University Medical Center, Durham, NC 27710, USATwo of the physiologically important processes thattake place in biological membranes are the partitioningof water-soluble proteins into the membrane and thesequestering of specific transmembrane proteins intomembrane microdomains or ‘rafts’. Although these twoprocesses often involve different classes of protein,recent biophysical studies indicate that they bothstrongly depend on the structural and elastic propertiesof the membrane bilayer. That is, both the partitioningof peptides into membranes and the distribution oftransmembrane peptides in the plane of the membraneare modulated by physical properties of the lipid bilayerthat are controlled by cholesterol content and the com-position 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. Lipidcontent varies according to the specific membranousorganelle; for example, sphingolipids and cholesterol areenriched in plasma membranes but nearly absent inendoplasmic reticulum membranes. Moreover, membranelipids differ widely in hydrocarbon chain composition, withvariations in hydrocarbon chain length and the number ofdouble bonds per chain.For cell biologists and membrane biochemists, a long-standing conundrum concerns the reasons why mem-branes contain so many types of lipid [1,2]. In terms ofprotein–lipid interactions and many biological functions,it is now clear that two distinct features of membranelipids are important: chemically specific properties ofthe individual lipid molecule, in particular those of theheadgroup; and structural and elastic (material) proper-ties of the whole bilayer (or microdomains in the bilayer),which are primarily determined by the composition of thelipid hydrocarbon chain.In biological processes, specific types of lipid moleculehave different roles [2]. Inositol and choline phospholipidsare sources of second messengers in transmembranesignaling [3,4]; phosphatidylcholine provides optimalfunctioning of 3-hydroxybutyrate dehydrogenase [5];cardiolipin supports mitochondrial inner membraneintegrity and proton conduction [6]; phosphatidylethano-lamine and phosphatidylglycerol enhance the activity ofa bacterial signal peptidase [7]; negatively chargedphospholipids bind peptides and proteins to the membranesurface [8]; and glycolipids interact with lectins and toxins,and are involved in cell–cell contacts [9].In addition, many studies have shown that the com-position of the hydrocarbon chain region of the bilayer isimportant in lipid–protein interactions and in modulatingmembrane functions. For example, the activities of specificmembrane pumps and transporters incorporated intobilayers depend on the length of the phospholipid acylchain [10,11]; and protein binding to membranes [12] andthe activity of enzymes and important membrane proteins,such as rhodopsin [13,14], are modulated by unsaturationin the phospholipid hydrocarbon chain. Moreover, specificmembrane proteins are sequestered into specialized regionsof plasma and Golgi membranes, called microdomains or‘rafts’, which have a more ordered hydrocarbon interiorthan the surrounding bilayer [15–17]. This sequestrationof some proteins into rafts, coupled with the exclusion ofothers [18], is thought to be essential to several membranefunctions, including signal transduction [18–21], mem-brane fusion [22] and protein trafficking [15,23].Here we discuss recent studies on the role of thestructural and mechanical properties of bilayers in twoaspects of peptide–lipid interactions: first, the partition-ing of physiologically important peptides from the aqueousphase into membranes; and second, the distribution oftransbilayer peptides in the plane of the bilayer and themechanisms involved in sorting peptides into or out ofmembrane rafts. Our particular focus is the role ofcholesterol in these peptide–lipid interactions, becausecholesterol modifies both the structural and the elasticproperties of bilayers. In short, cholesterol decreases thearea per phospholipid molecule [24], increases bilayerhydrophobic thickness [24] and increases the bilayer areacompressibility modulus (Ka), a measure of the cohesionbetween lipid molecules obtained from stress versus strainplots [25].Partitioning of water-soluble peptides into bilayersSeveral diverse classes of water-soluble protein andpeptide bind to both biological membranes and lipidbilayers [26–29]. Of such peptides, one of the bestcharacterized is melittin, a cationic peptide of 26 aminoacids from honeybee venom that induces channel for-mation in bilayers [30]. The interactions between melittinand bilayer vesicles have been analyzed by severaltechniques. Particularly useful for comparing bilayersystems are measurements of the partition coefficientand the resulting free energy of transfer (DGo) of melittinCorresponding author: Thomas J. McIntosh ([email protected]).Available online 6 May 2004Review TRENDS in Biochemical Sciences Vol.29 No.6 June 2004www.sciencedirect.com 0968-0004/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibs.2004.04.002from water to bilayer [31], and the melittin-inducedleakage of small water-soluble fluorescent probes encap-sulated in the vesicle interior [32,33]. Values of DGoprovide a quantitative measurement of the strength ofbinding (the more negative the DGovalue, the larger thebinding), whereas leakage data provide a functional assayfor this membrane-lytic peptide.Several studies have characterized melittin bindingand melittin-induced leakage for vesicles composed oflipids that are typical of eukaryotic membranes. Forexample, for electrically neutral bilayers comprisingpalmitoyloleoylphosphatidylcholine (POPC) or egg phos-phatidylcholine