Membrane Budding
2010; Cell Press; Volume: 143; Issue: 6 Linguagem: Inglês
10.1016/j.cell.2010.11.030
ISSN1097-4172
AutoresJames H. Hurley, Evžen Bouřa, Lars A. Carlson, Bartosz Różycki,
Tópico(s)Silk-based biomaterials and applications
ResumoMembrane budding is a key step in vesicular transport, multivesicular body biogenesis, and enveloped virus release. These events range from those that are primarily protein driven, such as the formation of coated vesicles, to those that are primarily lipid driven, such as microdomain-dependent biogenesis of multivesicular bodies. Other types of budding reside in the middle of this spectrum, including caveolae biogenesis, HIV-1 budding, and ESCRT-catalyzed multivesicular body formation. Some of these latter events involve budding away from cytosol, and this unusual topology involves unique mechanisms. This Review discusses progress toward understanding the structural and energetic bases of these different membrane-budding paradigms. Membrane budding is a key step in vesicular transport, multivesicular body biogenesis, and enveloped virus release. These events range from those that are primarily protein driven, such as the formation of coated vesicles, to those that are primarily lipid driven, such as microdomain-dependent biogenesis of multivesicular bodies. Other types of budding reside in the middle of this spectrum, including caveolae biogenesis, HIV-1 budding, and ESCRT-catalyzed multivesicular body formation. Some of these latter events involve budding away from cytosol, and this unusual topology involves unique mechanisms. This Review discusses progress toward understanding the structural and energetic bases of these different membrane-budding paradigms. Eukaryotic cells are defined by their compartmentalization into membrane-delimited structures. The protein and lipid content of these membranes is maintained and regulated by a constant flux of vesicular trafficking. Each vesicular trafficking event involves the budding of a membrane vesicle from a donor membrane, typically followed by its regulated transport to, docking to, and fusion with an acceptor membrane. Many viruses also have membrane envelopes and escape from host cells by membrane-budding events. Our laboratory has been characterizing the unusual membrane-budding reaction promoted by the ESCRTs, which has led us to take a fresh look at how membrane lipid properties might make protein-dependent, energetically expensive reactions easier. Several excellent reviews have covered the way proteins induce curvature in biological membranes (Farsad and De Camilli, 2003Farsad K. De Camilli P. Mechanisms of membrane deformation.Curr. Opin. Cell Biol. 2003; 15: 372-381Crossref PubMed Scopus (168) Google Scholar, McMahon and Gallop, 2005McMahon H.T. Gallop J.L. Membrane curvature and mechanisms of dynamic cell membrane remodelling.Nature. 2005; 438: 590-596Crossref PubMed Scopus (667) Google Scholar, Voeltz and Prinz, 2007Voeltz G.K. Prinz W.A. Sheets, ribbons and tubules - how organelles get their shape.Nat. Rev. Mol. Cell Biol. 2007; 8: 258-264Crossref PubMed Scopus (61) Google Scholar) and the physical principles of membrane curvature (Zimmerberg and Kozlov, 2006Zimmerberg J. Kozlov M.M. How proteins produce cellular membrane curvature.Nat. Rev. Mol. Cell Biol. 2006; 7: 9-19Crossref PubMed Scopus (431) Google Scholar). This Review will take a different viewpoint and consider the comparative roles of proteins and lipids in select examples of vesicular budding events (Figure 1) to discuss similarities and differences in budding events in synthetic versus cellular contexts, the potential roles of proteins in orchestrating lipid phase changes, and the roles of lipids in recruiting and regulating proteins. We also examine the implications of the above for cell physiology. This article is not intended as a comprehensive review of all cellular budding events. Rather, we consider emerging mechanistic thinking in multivesicular body formation and virus budding, placing these in the context of the classical mechanisms underlying budding of coated vesicles. The formation of spherical vesicles from a flat membrane of typical biological composition and no intrinsic propensity to curve entails a membrane-bending free energy (Helfrich, 1973Helfrich W. Elastic properties of lipid bilayers: theory and possible experiments.Z. Naturforsch. C. 1973; 28: 693-703Crossref PubMed Scopus (0) Google Scholar), ΔG = 8πκ ∼250–600 kBT, given κ ∼10–25 kBT, where kBT is thermal energy (Bloom et al., 1991Bloom M. Evans E. Mouritsen O.G. Physical properties of the fluid lipid-bilayer component of cell membranes: a perspective.Q. Rev. Biophys. 1991; 24: 293-397Crossref PubMed Google Scholar).This is important for biology because events that require thermal energy of this magnitude (that is, of ∼100 kBT or greater) do not occur spontaneously. Biophysical studies of membrane budding, which offer the promise of accounting for energetics, are typically carried out in vesicles that are much larger than their counterparts in biological systems. Fortunately, the energetic cost of bud formation is to a first approximation independent of the size of the bud. In pure lipid mixtures used in biophysical studies, vesicles are microns in size, spreading the energetic cost over ∼106 or more lipid molecules. In cells, however, membrane buds have a diameter of ∼20–100 nm, thus involving as few as 103–104 lipid molecules. This poses the question, how do a modest number of protein-lipid interactions create the free energy that is needed for budding, or alternatively, how do lipids themselves contribute to lowering the energy barrier? The dominant mechanism of membrane budding into the cytosol and the paradigm for protein-directed budding is the formation of coated vesicles (Figures 1F and 1G and Figure 2). Clathrin-coated vesicles (CCVs) are typically 60–100 nm in diameter (Bonifacino and Lippincott-Schwartz, 2003Bonifacino J.S. Lippincott-Schwartz J. Coat proteins: shaping membrane transport.Nat. Rev. Mol. Cell Biol. 2003; 4: 409-414Crossref PubMed Scopus (213) Google Scholar, Brodsky et al., 2001Brodsky F.M. Chen C.Y. Knuehl C. Towler M.C. Wakeham D.E. Biological basket weaving: formation and function of clathrin-coated vesicles.Annu. Rev. Cell Dev. Biol. 2001; 17: 517-568Crossref PubMed Scopus (422) Google Scholar). Clathrin can form baskets in vitro that resemble the CCVs in the absence of membranes, and the basket structure has been characterized in molecular detail (Fotin et al., 2004Fotin A. Cheng Y.F. Sliz P. Grigorieff N. Harrison S.C. Kirchhausen T. Walz T. Molecular model for a complete clathrin lattice from electron cryomicroscopy.Nature. 2004; 432: 573-579Crossref PubMed Scopus (217) Google Scholar). Clathrin itself binds neither membranes nor cargo but relies on adaptors for this function. Among the most comprehensively studied is adaptor protein complex 2 (AP-2 complex) (Robinson and Bonifacino, 2001Robinson M.S. Bonifacino J.S. Adaptor-related proteins.Curr. Opin. Cell Biol. 2001; 13: 444-453Crossref PubMed Scopus (352) Google Scholar), which functions in clathrin-mediated endocytosis at the plasma membrane. The AP-2 adaptor complex opens up in the presence of cargo and the lipid phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2) to form a flat platform capable of binding multiple PI(4,5)P2 and cargo molecules (Jackson et al., 2010Jackson L.P. Kelly B.T. McCoy A.J. Gaffry T. James L.C. Collins B.M. Höning S. Evans P.R. Owen D.J. A large-scale conformational change couples membrane recruitment to cargo binding in the AP2 clathrin adaptor complex.Cell. 2010; 141: 1220-1229Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The established role for PI(4,5)P2 in this pathway is to recruit AP-2 and other proteins to the site of budding. A role for PI(4,5)P2 clustering into microdomains has been suggested on theoretical grounds (Liu et al., 2006Liu J. Kaksonen M. Drubin D.G. Oster G. Endocytic vesicle scission by lipid phase boundary forces.Proc. Natl. Acad. Sci. USA. 2006; 103: 10277-10282Crossref PubMed Scopus (76) Google Scholar) but has yet to be directly visualized. (A) Structure of a clathrin basket from cytoelectron microscopy; reproduced by permission from Macmillan Publishers Ltd: Nature, Fotin et al., 2004Fotin A. Cheng Y.F. Sliz P. Grigorieff N. Harrison S.C. Kirchhausen T. Walz T. Molecular model for a complete clathrin lattice from electron cryomicroscopy.Nature. 2004; 432: 573-579Crossref PubMed Scopus (217) Google Scholar, copyright 2004. (B) COP II vesicles produced from purified components; reproduced by permission from Lee et al., 2005Lee M.C.S. Orci L. Hamamoto S. Futai E. Ravazzola M. Schekman R. Sar1p N-terminal helix initiates membrane curvature and completes the fission of a COPII vesicle.Cell. 2005; 122: 605-617Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar. (C) Structural parallels between clathrin, COP I, and COP II. Adapted from Lee and Goldberg, 2010Lee C. Goldberg J. Structure of coatomer cage proteins and the relationship among COPI, COPII, and clathrin vesicle coats.Cell. 2010; 142: 123-132Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar. Clathrin is absolutely required for the budding of AP-2- and cargo-rich plasma membrane domains, which remain flat in its absence (Hinrichsen et al., 2006Hinrichsen L. Meyerholz A. Groos S. Ungewickell E.J. Bending a membrane: how clathrin affects budding.Proc. Natl. Acad. Sci. USA. 2006; 103: 8715-8720Crossref PubMed Scopus (59) Google Scholar). However, clathrin monomers are flexible, which gives clathrin the ability to form different types of lattices and to adapt to various cargoes (Ehrlich et al., 2004Ehrlich M. Boll W. Van Oijen A. Hariharan R. Chandran K. Nibert M.L. Kirchhausen T. Endocytosis by random initiation and stabilization of clathrin-coated pits.Cell. 2004; 118: 591-605Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar). Given the flexibility of clathrin monomers, the energy of clathrin polymerization has been proposed on theoretical grounds to be insufficient on its own to bend the membrane into a bud (Nossal, 2001Nossal R. Energetics of clathrin basket assembly.Traffic. 2001; 2: 138-147Crossref PubMed Scopus (64) Google Scholar). However, this concept has yet to be confirmed experimentally and is not universally accepted. Cholesterol is important for clathrin-mediated endocytosis by many (though not all) accounts (Rodal et al., 1999Rodal S.K. Skretting G. Garred O. Vilhardt F. van Deurs B. Sandvig K. Extraction of cholesterol with methyl-beta-cyclodextrin perturbs formation of clathrin-coated endocytic vesicles.Mol. Biol. Cell. 1999; 10: 961-974Crossref PubMed Google Scholar, Subtil et al., 1999Subtil A. Gaidarov I. Kobylarz K. Lampson M.A. Keen J.H. McGraw T.E. Acute cholesterol depletion inhibits clathrin-coated pit budding.Proc. Natl. Acad. Sci. USA. 1999; 96: 6775-6780Crossref PubMed Scopus (357) Google Scholar), although it is less sensitive to cholesterol depletion than most coat-independent budding pathways (Sandvig et al., 2008Sandvig K. Torgersen M.L. Raa H.A. van Deurs B. Clathrin-independent endocytosis: from nonexisting to an extreme degree of complexity.Histochem. Cell Biol. 2008; 129: 267-276Crossref PubMed Scopus (83) Google Scholar). Clathrin, cargo adaptors, and PI(4,5)P2 are necessary but not sufficient on their own to induce membrane curvature. The essential early endocytic factor epsin wedges its amphipathic helix α0 into the membrane upon PI(4,5)P2 binding, promoting positive curvature (Ford et al., 2002Ford M.G.J. Mills I.G. Peter B.J. Vallis Y. Praefcke G.J.K. Evans P.R. McMahon H.T. Curvature of clathrin-coated pits driven by epsin.Nature. 2002; 419: 361-366Crossref PubMed Scopus (477) Google Scholar). The cargo-binding muniscin proteins FCHo1/2 (Syp1 in yeast) contain BAR domains that promote positive curvature very early in endocytosis (Henne et al., 2010Henne W.M. Boucrot E. Meinecke M. Evergren E. Vallis Y. Mittal R. McMahon H.T. FCHo proteins are nucleators of clathrin-mediated endocytosis.Science. 2010; 328: 1281-1284Crossref PubMed Scopus (121) Google Scholar, Reider et al., 2009Reider A. Barker S.L. Mishra S.K. Im Y.J. Maldonado-Báez L. Hurley J.H. Traub L.M. Wendland B. Syp1 is a conserved endocytic adaptor that contains domains involved in cargo selection and membrane tubulation.EMBO J. 2009; 28: 3103-3116Crossref PubMed Scopus (53) Google Scholar, Stimpson et al., 2009Stimpson H.E.M. Toret C.P. Cheng A.T. Pauly B.S. Drubin D.G. Early-arriving Syp1p and Ede1p function in endocytic site placement and formation in budding yeast.Mol. Biol. Cell. 2009; 20: 4640-4651Crossref PubMed Scopus (43) Google Scholar, Traub and Wendland, 2010Traub L.M. Wendland B. Cell biology: How to don a coat.Nature. 2010; 465: 556-557Crossref PubMed Scopus (4) Google Scholar). In principle, the reagents and concepts would appear to be in place to reconstitute clathrin-dependent membrane budding. Reconstitution of clathrin-mediated endocytosis using synthetic lipids and purified proteins would be an important step in determining whether clathrin, AP-2, one or more amphipathic helix and/or BAR domain proteins, and PI(4,5)P2 constitute the minimum requirements for membrane bud formation in this pathway. The scission of the clathrin-coated bud to form a detached vesicle is a complex process in its own right, and the reader is referred to recent reviews (Pucadyil and Schmid, 2009Pucadyil T.J. Schmid S.L. Conserved functions of membrane active GTPases in coated vesicle formation.Science. 2009; 325: 1217-1220Crossref PubMed Scopus (89) Google Scholar). Finally, following scission, the clathrin coat is removed by the ATP-dependent action of the molecular chaperone Hsc70 and its cofactor auxillin (Eisenberg and Greene, 2007Eisenberg E. Greene L.E. Multiple roles of auxilin and hsc70 in clathrin-mediated endocytosis.Traffic. 2007; 8: 640-646Crossref PubMed Scopus (78) Google Scholar). It is only following nucleotide hydrolysis that the energetic cost of clathrin-induced membrane deformation is finally paid, making the full reaction cycle—from flat membrane to uncoated vesicle—thermodynamically irreversible. Vesicles carrying cargo from the endoplasmic reticulum (ER) to the Golgi are coated by the COP II complex, which, like clathrin, can form membrane-free baskets in vitro with vesicle-like dimensions (Stagg et al., 2006Stagg S.M. Gürkan C. Fowler D.M. LaPointe P. Foss T.R. Potter C.S. Carragher B. Balch W.E. Structure of the Sec13/31 COPII coat cage.Nature. 2006; 439: 234-238Crossref PubMed Scopus (139) Google Scholar). COP II vesicles have a preferred size, but as with clathrin, the flexibility of the COP II subunits allows formation of expanded lattices that can accommodate large cargoes such as procollagen and large lipoprotein particles known as chylomicrons (Stagg et al., 2008Stagg S.M. LaPointe P. Razvi A. Gürkan C. Potter C.S. Carragher B. Balch W.E. Structural basis for cargo regulation of COPII coat assembly.Cell. 2008; 134: 474-484Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). COP II vesicle budding has been reconstituted in vitro from purified proteins and synthetic lipids (Lee et al., 2005Lee M.C.S. Orci L. Hamamoto S. Futai E. Ravazzola M. Schekman R. Sar1p N-terminal helix initiates membrane curvature and completes the fission of a COPII vesicle.Cell. 2005; 122: 605-617Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, Matsuoka et al., 1998Matsuoka K. Orci L. Amherdt M. Bednarek S.Y. Hamamoto S. Schekman R. Yeung T. COPII-coated vesicle formation reconstituted with purified coat proteins and chemically defined liposomes.Cell. 1998; 93: 263-275Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar). A membrane consisting only of synthetic unsaturated phospholipids was capable of supporting budding (Matsuoka et al., 1998Matsuoka K. Orci L. Amherdt M. Bednarek S.Y. Hamamoto S. Schekman R. Yeung T. COPII-coated vesicle formation reconstituted with purified coat proteins and chemically defined liposomes.Cell. 1998; 93: 263-275Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar). COP II consists of the Sec23/24 subcomplex, which binds lipids and cargo via a gently curved face (Bi et al., 2002Bi X. Corpina R.A. Goldberg J. Structure of the Sec23/24-Sar1 pre-budding complex of the COPII vesicle coat.Nature. 2002; 419: 271-277Crossref PubMed Scopus (218) Google Scholar), the Sec13/31 subcomplex, which forms an outer cage around the vesicle, and the membrane-bending GTPase Sar1. The Sec23/24 and Sec13/31 subcomplexes in combination are sufficient to form buds, with Sar1 strictly required only for the scission of the buds. GTP hydrolysis by Sar1 provides energy input into the system, making the overall process (which culminates in the uncoating of cargo-loaded vesicles) thermodynamically irreversible. COP I-coated vesicles are responsible for retrograde traffic from the Golgi to the ER, and this reaction has also been reconstituted from purified proteins and synthetic lipids. The budding reaction requires the coatomer complex, GTP-bound Arf1, and protein cargo tails tethered to the membrane but has no special lipid requirements (Bremser et al., 1999Bremser M. Nickel W. Schweikert M. Ravazzola M. Amherdt M. Hughes C.A. Söllner T.H. Rothman J.E. Wieland F.T. Coupling of coat assembly and vesicle budding to packaging of putative cargo receptors.Cell. 1999; 96: 495-506Abstract Full Text Full Text PDF PubMed Google Scholar). Budding occurs even from vesicles composed of the pure synthetic phospholipid DOPC doped with small amounts of a lipopeptide cargo. Recently, a composite crystallographic structure of cage-forming components of coatomer consisting of the α, β′, and ɛ subunits has been determined and shown to resemble the clathrin triskelion (Lee and Goldberg, 2010Lee C. Goldberg J. Structure of coatomer cage proteins and the relationship among COPI, COPII, and clathrin vesicle coats.Cell. 2010; 142: 123-132Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). In sum, COP I and COP II provide some of the purest examples of protein-directed membrane budding, in which the protein coat imposes its shape upon the membrane with minimal dependence on its lipid composition. In contrast to the protein-dominated paradigm of coated vesicle budding, phase separation in simple lipid mixtures can drive budding on a micron scale in synthetic model membranes, in the absence of proteins (Baumgart et al., 2003Baumgart T. Hess S.T. Webb W.W. Imaging coexisting fluid domains in biomembrane models coupling curvature and line tension.Nature. 2003; 425: 821-824Crossref PubMed Scopus (728) Google Scholar) (Figure 1A and Figure 3). Membrane bilayers can adopt either a solid or a liquid phase, with the translational and conformational order of the lipid chains depending on their composition and the temperature. The liquid phase is the more relevant to biology and can be subdivided into liquid disordered (Ld) and liquid ordered (Lo) phases. Lipids in the Ld phase have higher conformational freedom and diffusion coefficients than in the Lo phase. At biological temperatures, the Ld and Lo phases can coexist in membranes of mixed composition (Elson et al., 2010Elson E.L. Fried E. Dolbow J.E. Genin G.M. Phase separation in biological membranes: Integration of theory and experiment.Annu. Rev. Biophys. 2010; 39: 207-226Crossref PubMed Scopus (51) Google Scholar, García-Sáez and Schwille, 2010García-Sáez A.J. Schwille P. Stability of lipid domains.FEBS Lett. 2010; 584: 1653-1658Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). (A) Coexistence of phases in model membranes visualized by atomic force microscopy in a supported bilayer (a membrane bilayer adsorbed onto a solid support, usually glass). Reproduced with permission from Chiantia et al., 2006Chiantia S. Kahya N. Ries J. Schwille P. Effects of ceramide on liquid-ordered domains investigated by simultaneous AFM and FCS.Biophys. J. 2006; 90: 4500-4508Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar. (B) Phase transitions in a single-lipid membrane analyzed by molecular dynamics simulations. Reproduced with permission from Heller et al., 1993Heller H. Schaefer M. Schulten K. Molecular dynamics simulation of a bilayer of 200 lipids in the gel and in the liquid crystal phases.J. Phys. Chem. 1993; 97: 8343-8360Crossref Google Scholar. Copyright 1993 American Chemical Society. (C) Schematic model of a raft-type membrane microdomain, including a model of a myristoylated ESCRT-III subunit Vps20 as an example of protein that might anchor to rafts. In general, phospholipids with unsaturated chains prefer the Ld phase, whereas cholesterol, sphingolipids, and phospholipids with saturated chains prefer the Lo phase (Lingwood and Simons, 2010Lingwood D. Simons K. Lipid rafts as a membrane-organizing principle.Science. 2010; 327: 46-50Crossref PubMed Scopus (932) Google Scholar). Typically, the energetic cost for contact between dissimilar lipids is small, ∼0.5 kBT (García-Sáez and Schwille, 2010García-Sáez A.J. Schwille P. Stability of lipid domains.FEBS Lett. 2010; 584: 1653-1658Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar), but becomes significant when summed over many lipids. The higher acyl chain order in the Lo phase results in their elongation to their maximum extent, hence Lo membrane domains are thicker than Ld domains. The height mismatch at the phase boundary is energetically unfavorable because it forces the polar headgroup region of the Ld domain into contact with the hydrophobic portion of the Lo domain. The free energy cost per unit length is known as the line tension and has units of force. In order to minimize the free energy associated with line tension, membrane domains will coalesce with one another into circular zones. When circular domains reach a critical size at which the line tension energy term exceeds the Helfrich (curvature-dependent) energy of membrane deformation, the membrane will deform out of plane in order to minimize the zone of contact (Lipowsky, 1992Lipowsky R. Budding of membranes induced by intramembrane domains.J. Phys. II France. 1992; 2: 1825-1840Crossref Google Scholar). If the line tension is high enough, the neck connecting the membrane bud can be severed, leading to the formation of detached vesicles. In addition to line tension effects, membrane microdomain formation can bend membranes by concentrating lipids with distinct intrinsic curvatures, and the contents of such microdomains can not only drive budding but dictate its direction (Bacia et al., 2005Bacia K. Schwille P. Kurzchalia T. Sterol structure determines the separation of phases and the curvature of the liquid-ordered phase in model membranes.Proc. Natl. Acad. Sci. USA. 2005; 102: 3272-3277Crossref PubMed Scopus (196) Google Scholar). The complex lipid mixture of the plasma membrane supports phase separation in micron-sized domains when reconstituted in giant unilamellar vesicles (Baumgart et al., 2007Baumgart T. Hammond A.T. Sengupta P. Hess S.T. Holowka D.A. Baird B.A. Webb W.W. Large-scale fluid/fluid phase separation of proteins and lipids in giant plasma membrane vesicles.Proc. Natl. Acad. Sci. USA. 2007; 104: 3165-3170Crossref PubMed Scopus (233) Google Scholar). However, in living cells, membrane microdomains are heterogeneous, highly dynamic nanoscale structures (Hancock, 2006Hancock J.F. Lipid rafts: contentious only from simplistic standpoints.Nat. Rev. Mol. Cell Biol. 2006; 7: 456-462Crossref PubMed Scopus (454) Google Scholar, Lingwood and Simons, 2010Lingwood D. Simons K. Lipid rafts as a membrane-organizing principle.Science. 2010; 327: 46-50Crossref PubMed Scopus (932) Google Scholar, Pike, 2006Pike L.J. Rafts defined: a report on the Keystone Symposium on Lipid Rafts and Cell Function.J. Lipid Res. 2006; 47: 1597-1598Crossref PubMed Scopus (510) Google Scholar). In the most up-to-date biophysical view, these nanoscale structures likely correspond to critical fluctuations (Veatch et al., 2007Veatch S.L. Soubias O. Keller S.L. Gawrisch K. Critical fluctuations in domain-forming lipid mixtures.Proc. Natl. Acad. Sci. USA. 2007; 104: 17650-17655Crossref PubMed Scopus (144) Google Scholar). Although the concepts of the Lo and Ld phases are oversimplifications of the variety of dynamic membrane substructures that exist in cells (Lingwood and Simons, 2010Lingwood D. Simons K. Lipid rafts as a membrane-organizing principle.Science. 2010; 327: 46-50Crossref PubMed Scopus (932) Google Scholar), they will be used in this Review because they are useful intuitive handles, deeply ingrained in the literature, and helpful in relating model membrane studies to biology. Most, but not all, of the membrane microdomains implicated in cellular budding are the sterol- and sphingolipid-rich domains known as “rafts.” Why don't rafts and other microdomains coalesce on the micron scale in living cells, as they do in model membranes? The answer is not known, but the action of the cytoskeleton and membrane traffic, and the large fraction of protein in cellular membranes, are usually invoked. Indeed, it is to be expected that cells would have mechanisms to block the unchecked growth of microdomains, as the ensuing spontaneous vesiculation of cell membranes would be disastrous. Soluble and lumenally anchored cargoes, viruses, and toxins are selectively transported in vesicular carriers even though they have no direct communication with the cytosol to signal their packaging and sorting. In some cases, transmembrane-sorting receptors serve as adaptors to link cargo to conventional cytosolic coat complexes. In other cases, membrane rafts make the link. Simian virus 40 (SV40) and cholera toxin enter cells by binding to multiple molecules of the ganglioside GM1 (Damm et al., 2005Damm E.M. Pelkmans L. Kartenbeck J. Mezzacasa A. Kurzchalia T. Helenius A. Clathrin- and caveolin-1-independent endocytosis: entry of simian virus 40 into cells devoid of caveolae.J. Cell Biol. 2005; 168: 477-488Crossref PubMed Scopus (239) Google Scholar, Kirkham et al., 2005Kirkham M. Fujita A. Chadda R. Nixon S.J. Kurzchalia T.V. Sharma D.K. Pagano R.E. Hancock J.F. Mayor S. Parton R.G. Ultrastructural identification of uncoated caveolin-independent early endocytic vehicles.J. Cell Biol. 2005; 168: 465-476Crossref PubMed Scopus (201) Google Scholar), a raft-favoring lipid. The cholera toxin B subunit (Merritt et al., 1994Merritt E.A. Sarfaty S. van den Akker F. L'Hoir C. Martial J.A. Hol W.G.J. Crystal structure of cholera toxin B-pentamer bound to receptor GM1 pentasaccharide.Protein Sci. 1994; 3: 166-175Crossref PubMed Google Scholar) and the SV40 VP1 protein (Neu et al., 2008Neu U. Woellner K. Gauglitz G. Stehle T. Structural basis of GM1 ganglioside recognition by simian virus 40.Proc. Natl. Acad. Sci. USA. 2008; 105: 5219-5224Crossref PubMed Scopus (72) Google Scholar) both bind to GM1 as pentamers. Cholera toxin pentamer binds GM1 (Figure 4) and thus induces formation of an Lo microdomain in model membranes (Hammond et al., 2005Hammond A.T. Heberle F.A. Baumgart T. Holowka D. Baird B. Feigenson G.W. Crosslinking a lipid raft component triggers liquid ordered-liquid disordered phase separation in model plasma membranes.Proc. Natl. Acad. Sci. USA. 2005; 102: 6320-6325Crossref PubMed Scopus (147) Google Scholar) and in turn leads to budding (Bacia et al., 2005Bacia K. Schwille P. Kurzchalia T. Sterol structure determines the separation of phases and the curvature of the liquid-ordered phase in model membranes.Proc. Natl. Acad. Sci. USA. 2005; 102: 3272-3277Crossref PubMed Scopus (196) Google Scholar, Ewers et al., 2010Ewers H. Römer W. Smith A.E. Bacia K. Dmitrieff S. Chai W.G. Mancini R. Kartenbeck J. Chambon V. Berland L. et al.GM1 structure determines SV40-induced membrane invagination and infection.Nat. Cell Biol. 2010; 12 (1–12): 11-18Crossref PubMed Scopus (96) Google Scholar). Shiga toxin B subunit binds the glycolipid Gb3 and appears to operate by a similar paradigm. In this case tubular vesicles are formed, and lipid compression favoring negative curvature is thought to be the driving force (Römer et al., 2007Römer W. Berland L. Chambon V. Gaus K. Windschiegl B. Tenza D. Aly M.R.E. Fraisier V. Florent J.C. Perrais D. et al.Shiga toxin induces tubular membrane invaginations for its uptake into cells.Nature. 2007; 450: 670-675Crossref PubMed Scopus (203) Google Scholar). In each of these examples, it is clear that clustering of lipids leads to important changes in membrane structure that contribute to budding. The proposed physical mechanisms remain speculative, however. Revealing these mechanisms remains a profound challenge to experimentalists and thus is an area that will benefit from increasing sophisticated computer simulations of membrane dynamics on realistic timescales. (A) Simian virus 40 VP1 pentamer bound to the membrane via the headgroup of the ganglioside GM1 (Neu et al., 2008Neu U. Woellner K. Gauglitz G. Stehle T. Structural basis of GM1 ganglioside recognition by simian virus 40.Proc. Natl. Acad. Sci. USA. 2008; 105: 5219-5224Crossref PubMed Scopus (72) Google Scholar). (B) Cholera toxin B subunit pentamer bound to GM1 (Merritt et al., 1994Merritt E.A. Sarfaty S. van den Akker F. L'Hoir C. Martial J.A. Hol W.G.J. Crystal structure of cholera toxin B-pentamer bound to receptor GM1 pentasaccharide.Protein Sci. 1994; 3: 166-175Crossref PubMed Google Scholar). (C) Composite model of the myristoylated HIV-1 matrix domain trimer bound to PI(4,5)P2 (Hill et al., 1996Hill C.P. Worthylake D. Bancroft D.P. Christensen A.M. Sundquist W.I. Crystal structures of the trimeric human immunodeficiency virus type 1 matrix protein: implications for membrane association and assembly.Proc. Natl. Acad. Sci. USA. 1996; 93: 3099-3104Crossref PubMed Scopus (292) Google Scholar, Saad et al., 2006Saad J.S. Miller J. Tai J. Kim A. Ghanam R.H. Summers M.F. 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