Protein-Mediated Transformation of Lipid Vesicles into Tubular Networks

Key cellular processes are frequently accompanied by protein-facilitated shape changes in the plasma membrane. N-BAR-domain protein modules generate curvature by means of complex interactions with the membrane surface. The way they assemble and the mechanism by which they operate are largely dependent on their binding density. Although the mechanism at lower densities has recently begun to emerge, how membrane scaffolds form at high densities remains unclear. By combining electron microscopy and multiscale simulations, we show that N-BAR proteins at high densities can transform a lipid vesicle into a 3D tubular network. We show that this process is a consequence of excess adhesive energy combined with the local stiffening of the membrane, which occurs in a narrow range of mechanical properties of both the membrane and the protein. We show that lipid diffusion is significantly reduced by protein binding at this density regime and even more in areas of high Gaussian curvature, indicating a potential effect on molecular transport in cells. Finally, we reveal that the breaking of the bilayer topology is accompanied by the nematic arrangement of the protein on the surface, a structural motif that likely drives the formation of reticular structures in living cells.     

SNARE-mediated lipid mixing depends on the physical state of the vesicles

Reconstitution experiments have suggested that N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins constitute a minimal membrane fusion machinery but have yielded contradictory results, and it is unclear whether the mechanism of membrane merger is related to the stalk mechanism that underlies physiological membrane fusion. Here we show that reconstitution of solubilized neuronal SNAREs into preformed 100 nm liposomes (direct method) yields proteoliposomes with more homogeneous sizes and protein densities than the standard reconstitution method involving detergent cosolubilization of proteins and lipids. Standard reconstitutions yield slow but efficient lipid mixing at high protein densities and variable amounts of lipid mixing at moderate protein densities. However, the larger, more homogenous proteoliposomes prepared by the direct method yield almost no lipid mixing at moderate protein densities. These results suggest that the lipid mixing observed for standard reconstitutions is dominated by the physical state of the membrane, perhaps due to populations of small vesicles (or micelles) with high protein densities and curvature stress created upon reconstitution. Accordingly, changing membrane spontaneous curvature by adding lysophospholipids inhibits the lipid mixing observed for standard reconstitutions. Our data indicate that the lipid mixing caused by high SNARE densities and/or curvature stress occurs by a stalk mechanism resembling the mechanism of fusion between biological membranes, but the neuronal SNAREs are largely unable to induce lipid mixing at physiological protein densities and limited curvature stress.     

Membrane bending by protein-protein crowding

Curved membranes are an essential feature of dynamic cellular structures, including endocytic pits, filopodia protrusions and most organelles. It has been proposed that specialized proteins induce curvature by binding to membranes through two primary mechanisms: membrane scaffolding by curved proteins or complexes; and insertion of wedge-like amphipathic helices into the membrane. Recent computational studies have raised questions about the efficiency of the helix-insertion mechanism, predicting that proteins must cover nearly 100% of the membrane surface to generate high curvature, an improbable physiological situation. Thus, at present, we lack a sufficient physical explanation of how protein attachment bends membranes efficiently. On the basis of studies of epsin1 and AP180, proteins involved in clathrin-mediated endocytosis, we propose a third general mechanism for bending fluid cellular membranes: protein-protein crowding. By correlating membrane tubulation with measurements of protein densities on membrane surfaces, we demonstrate that lateral pressure generated by collisions between bound proteins drives bending. Whether proteins attach by inserting a helix or by binding lipid heads with an engineered tag, protein coverage above ~20% is sufficient to bend membranes. Consistent with this crowding mechanism, we find that even proteins unrelated to membrane curvature, such as green fluorescent protein (GFP), can bend membranes when sufficiently concentrated. These findings demonstrate a highly efficient mechanism by which the crowded protein environment on the surface of cellular membranes can contribute to membrane shape change.     

Lipid rafts act as specialized domains for tetanus toxin binding and internalization into neurons

Tetanus (TeNT) is a zinc protease that blocks neurotransmission by cleaving the synaptic protein vesicle-associated membrane protein/synaptobrevin. Although its intracellular catalytic activity is well established, the mechanism by which this neurotoxin interacts with the neuronal surface is not known. In this study, we characterize p15s, the first plasma membrane TeNT binding proteins and we show that they are glycosylphosphatidylinositol-anchored glycoproteins in nerve growth factor (NGF)-differentiated PC12 cells, spinal cord cells, and purified motor neurons. We identify p15 as neuronal Thy-1 in NGF-differentiated PC12 cells. Fluorescence lifetime imaging microscopy measurements confirm the close association of the binding domain of TeNT and Thy-1 at the plasma membrane. We find that TeNT is recruited to detergent-insoluble lipid microdomains on the surface of neuronal cells. Finally, we show that cholesterol depletion affects a raft subpool and blocks the internalization and intracellular activity of the toxin. Our results indicate that TeNT interacts with target cells by binding to lipid rafts and that cholesterol is required for TeNT internalization and/or trafficking in neurons.     

BAR domains, amphipathic helices and membrane-anchored proteins use the same mechanism to sense membrane curvature

The internal membranes of eukaryotic cells are all twists and bends characterized by high curvature. During recent years it has become clear that specific proteins sustain these curvatures while others simply recognize membrane shape and use it as “molecular information” to organize cellular processes in space and time. Here we discuss this new important recognition process termed membrane curvature sensing (MCS). First, we review a new fluorescence-based experimental method that allows characterization of MCS using measurements on single vesicles and compare it to sensing assays that use bulk/ensemble liposome samples of different mean diameter. Next, we describe two different MCS protein motifs (amphipathic helices and BAR domains) and suggest that in both cases curvature sensitive membrane binding results from asymmetric insertion of hydrophobic amino acids in the lipid membrane. This mechanism can be extended to include the insertion of alkyl chain in the lipid membrane and consequently palmitoylated and myristoylated proteins are predicted to display similar curvature sensitive binding. Surprisingly, in all the aforementioned cases, MCS is predominantly mediated by a higher density of binding sites on curved membranes instead of higher affinity as assumed so far. Finally, we integrate these new insights into the debate about which motifs are involved in sensing versus induction of membrane curvature and what role MCS proteins may play in biology.     

Membrane Fusion Promoted by Increasing Surface Densities of the Paramyxovirus F and HN Proteins: Comparison of Fusion Reactions Mediated by Simian Virus 5 F, Human Parainfluenza Virus Type 3 F, and Influenza Virus HA

The membrane fusion reaction promoted by the paramyxovirus simian virus 5 (SV5) and human parainfluenza virus type 3 (HPIV-3) fusion (F) proteins and hemagglutinin-neuraminidase (HN) proteins was characterized when the surface densities of F and HN were varied. Using a quantitative content mixing assay, it was found that the extent of SV5 F-mediated fusion was dependent on the surface density of the SV5 F protein but independent of the density of SV5 HN protein, indicating that HN serves only a binding function in the reaction. However, the extent of HPIV-3 F protein promoted fusion reaction was found to be dependent on surface density of HPIV-3 HN protein, suggesting that the HPIV-3 HN protein is a direct participant in the fusion reaction. Analysis of the kinetics of lipid mixing demonstrated that both initial rates and final extents of fusion increased with rising SV5 F protein surface densities, suggesting that multiple fusion pores can be active during SV5 F protein-promoted membrane fusion. Initial rates and extent of lipid mixing were also found to increase with increasing influenza virus hemagglutinin protein surface density, suggesting parallels between the mechanism of fusion promoted by these two viral fusion proteins.     

Lipid hapten containing membrane targets can trigger specific immunoglobulin E-dependent degranulation of rat basophil leukemia cells

We have studied the binding of liposomes containing dinitrophenylated lipid to rat basophil leukemia cells armed with monoclonal anti-dinitrophenyl IgE. The liposomes were either "fluid" at 37 degrees C (dimyristoylphosphatidylcholine or an equimolar binary mixture dipalmitoylphosphatidylcholine and cholesterol) or "solid" (dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, or dibehanoylphosphatidylcholine). We have also studied the immune mediated degranulation of these cells induced by the above lipid membrane targets. In some cases both studies were carried out with liposomes containing various surface densities of lipid haptens. From these studies we conclude that freely mobile nonaggregated lipid haptens in bilayer membrane targets can trigger efficient serotonin release from rat basophil leukemia cells in the presence of specific antihapten IgE. Solid target membranes are also effective as stimulators of serotonin release. The release of serotonin depends strongly on the surface density of lipid haptens over a narrow range of surface densities. These studies with lipid membrane targets having well defined physical properties indicate the need for generalized molecular models of receptor-mediated cell triggering.     

Sensing Membrane Stresses by Protein Insertions

Protein domains shallowly inserting into the membrane matrix are ubiquitous in peripheral membrane proteins involved in various processes of intracellular membrane shaping and remodeling. It has been suggested that these domains sense membrane curvature through their preferable binding to strongly curved membranes, the binding mechanism being mediated by lipid packing defects. Here we make an alternative statement that shallow protein insertions are universal sensors of the intra-membrane stresses existing in the region of the insertion embedding rather than sensors of the curvature per se. We substantiate this proposal computationally by considering different independent ways of the membrane stress generation among which some include changes of the membrane curvature whereas others do not alter the membrane shape. Our computations show that the membrane-binding coefficient of shallow protein insertions is determined by the resultant stress independently of the way this stress has been produced. By contrast, consideration of the correlation between the insertion binding and the membrane curvature demonstrates that the binding coefficient either increases or decreases with curvature depending on the factors leading to the curvature generation. To validate our computational model, we treat quantitatively the experimental results on membrane binding by ALPS1 and ALPS2 motifs of ArfGAP1.     

Lipid demixing and protein-protein interactions in the adsorption of charged proteins on mixed membranes

The adsorption free energy of charged proteins on mixed membranes, containing varying amounts of (oppositely) charged lipids, is calculated based on a mean-field free energy expression that accounts explicitly for the ability of the lipids to demix locally, and for lateral interactions between the adsorbed proteins. Minimization of this free energy functional yields the familiar nonlinear Poisson-Boltzmann equation and the boundary condition at the membrane surface that allows for lipid charge rearrangement. These two self-consistent equations are solved simultaneously. The proteins are modeled as uniformly charged spheres and the (bare) membrane as an ideal two-dimensional binary mixture of charged and neutral lipids. Substantial variations in the lipid charge density profiles are found when highly charged proteins adsorb on weakly charged membranes; the lipids, at a certain demixing entropy penalty, adjust their concentration in the vicinity of the adsorbed protein to achieve optimal charge matching. Lateral repulsive interactions between the adsorbed proteins affect the lipid modulation profile and, at high densities, result in substantial lowering of the binding energy. Adsorption isotherms demonstrating the importance of lipid mobility and protein-protein interactions are calculated using an adsorption equation with a coverage-dependent binding constant. Typically, at bulk-surface equilibrium (i.e., when the membrane surface is "saturated" by adsorbed proteins), the membrane charges are "overcompensated" by the protein charges, because only about half of the protein charges (those on the hemispheres facing the membrane) are involved in charge neutralization. Finally, it is argued that the formation of lipid-protein domains may be enhanced by electrostatic adsorption of proteins, but its origin (e.g., elastic deformations associated with lipid demixing) is not purely electrostatic.     

Neutron scattering studies of photosynthetic membranes in aqueous dispersion

A method for structural analysis of biological membranes using neutron scattering from suspensions is described and applied to photosynthetic membranes from bacteria. The variation of scattering density across the membrane is analysed using small-angle scattering and contrast variation with H₂O/²H₂O mixtures. Effects due to membrane curvature and scattering density variation in the plane of the membrane are evaluated. Thickness parameters (D) are obtained from the small-angle scattering data, which are the one-dimensional analogues of radii of gyration. The formalism of contrast variation is used to describe the change of intensity and thickness parameter with H₂O/²H₂O mixture. The results are expressed in terms of a thickness parameter at infinite contrast, which is directly related to the physical thickness of the membrane, and a measure of the variation of the scattering density across the membrane, produced, for example, by the higher scattering densities of the polar surfaces relative to a hydrocarbon interior of the membrane. Asymmetry in the membrane scattering density is also evaluated.The results for photosynthetic membranes demonstrate a lipid hydrocarbon core in the membrane. About two-thirds of the protein is closely associated with the lipid layer, and no substantial amounts of protein project more than short distances from the lipid layer. There is a contribution to the variation in scattering density across the membrane that cannot be attributed to lipid, and may involve scattering density heterogeneity within the protein, giving a high proportion of hydrophobic protein segments at the interior of the membrane that have lower scattering densities than the hydrophilic segments at the surfaces of the membrane. The membrane scattering density is not markedly asymmetric. Several alternative structures previously proposed for photosynthetic membranes are incompatible with these results.     

Probucol inactivates ABCA1 in the plasma membrane with respect to its mediation of apolipoprotein binding and high density lipoprotein assembly and to its proteolytic degradation

Probucol has been shown to inhibit the release of cellular lipid by helical apolipoprotein and thereby to reduce plasma high density lipoprotein. We attempted to explore the underlying mechanism for this effect in human fibroblast WI-38. Probucol inhibited the apoA-I-mediated cellular lipid release and binding of apoA-I to the cells in a dose-dependent manner. It did not influence cellular uptake of low density lipoprotein, transport of cholesterol to the cell surface whether de novo synthesized or delivered as low density lipoprotein, and overall cellular content of cholesterol, although biosynthesis of lipids from acetate was somewhat increased. Probucol did not affect the mRNA level of ABCA1, and ABCA1 was recovered along with marker proteins for plasma membrane regardless of the presence of probucol. However, the protein level of ABCA1 increased, and the rate of its decay in the presence of cycloheximide was slower in the probucol-treated cells. ABCA1 in the probucol-treated cells was resistant to digestion by calpain but not by trypsin. We concluded that probucol inactivates ABCA1 in the plasma membrane with respect to its function in mediating binding of and lipid release by apolipoprotein and with respect to proteolytic degradation by calpain.     

Nonlinear sorting, curvature generation, and crowding of endophilin N-BAR on tubular membranes

The curvature of biological membranes is controlled by membrane-bound proteins. For example, during endocytosis, the sorting of membrane components, vesicle budding, and fission from the plasma membrane are mediated by adaptor and accessory proteins. Endophilin is a peripherally binding membrane protein that functions as an endocytic accessory protein. Endophilin's membrane tubulation capacity is well known. However, to understand the thermodynamic and mechanical aspects of endophilin function, experimental measurements need to be compared to quantitative theoretical models. We present measurements of curvature sorting and curvature generation of the endophilin A1 N-BAR domain on tubular membranes pulled from giant unilamellar vesicles. At low concentration, endophilin functions primarily as a membrane curvature sensor; at high concentrations, it also generates curvature. We determine the spontaneous curvature induced by endophilin and observe sigmoidal curvature/composition coupling isotherms that saturate at high membrane tensions and protein solution concentrations. The observation of saturation is supported by a strong dependence of lateral diffusion coefficients on protein density on the tether membrane. We develop a nonlinear curvature/composition coupling model that captures our experimental observations. Our model predicts a curvature-induced phase transition among two states with varying protein density and membrane curvature. This transition could act as a switch during endocytosis.     

The interaction of viral fusion peptides with lipid membranes

In this paper, we studied fusogenic peptides of class I-III fusion proteins, which are relevant to membrane fusion for certain enveloped viruses, in contact with model lipid membranes. We resolved the vertical structure and examined the adsorption or penetration behavior of the fusogenic peptides at phospholipid Langmuir monolayers with different initial surface pressures with x-ray reflectometry. We show that the fusion loops of tick-borne encephalitis virus (TBEV) glycoprotein E and vesicular stomatitis virus (VSV) G-protein are not able to insert deeply into model lipid membranes, as they adsorbed mainly underneath the headgroups with only limited penetration depths into the lipid films. In contrast, we observed that the hemagglutinin 2 fusion peptide (HA2-FP) and the VSV-transmembrane domain (VSV-TMD) can penetrate deeply into the membranes. However, in the case of VSV-TMD, the penetration was suppressed already at low surface pressures, whereas HA2-FP was able to insert even into highly compressed films. Membrane fusion is accompanied by drastic changes of the membrane curvature. To investigate how the peptides affect the curvature of model lipid membranes, we examined the effect of the fusogenic peptides on the equilibration of cubic monoolein structures after a phase transition from a lamellar state induced by an abrupt hydrostatic pressure reduction. We monitored this process in presence and absence of the peptides with small-angle x-ray scattering and found that HA2-FP and VSV-TMD drastically accelerate the equilibration, while the fusion loops of TBEV and VSV stabilize the swollen state of the lipid structures. In this work, we show that the class I fusion peptide of HA2 penetrates deeply into the hydrophobic region of membranes and is able to promote and accelerate the formation of negative curvature. In contrast, we found that the class II and III fusion loops of TBEV and VSV tend to counteract negative membrane curvature.     

Mechanism of ATP-binding cassette transporter A1-mediated cellular lipid efflux to apolipoprotein A-I and formation of high density lipoprotein particles

The ATP-binding cassette transporter A1 (ABCA1) plays a critical role in the biogenesis of high density lipoprotein (HDL) particles and in mediating cellular cholesterol efflux. The mechanism by which ABCA1 achieves these effects is not established, despite extensive investigation. Here, we present a model that explains the essential features, especially the effects of ABCA1 activity in inducing apolipoprotein (apo) A-I binding to cells and the compositions of the discoidal HDL particles that are produced. The apo A-I/ABCA1 reaction scheme involves three steps. First, there is binding of a small regulatory pool of apo A-I to ABCA1, thereby enhancing net phospholipid translocation to the plasma membrane exofacial leaflet; this leads to unequal lateral packing densities in the two leaflets of the phospholipid bilayer. Second, the resultant membrane strain is relieved by bending and by creation of exovesiculated lipid domains. The formation of highly curved membrane surface promotes high affinity binding of apo A-I to these domains. Third, this pool of bound apo A-I spontaneously solubilizes the exovesiculated domain to create discoidal nascent HDL particles. These particles contain two, three, or four molecules of apo A-I and a complement of membrane phospholipid classes together with some cholesterol. A key feature of this mechanism is that membrane bending induced by ABCA1 lipid translocase activity creates the conditions required for nascent HDL assembly by apo A-I. Overall, this mechanism is consistent with the known properties of ABCA1 and apo A-I and reconciles many of the apparently discrepant findings in the literature.     

FisB relies on homo-oligomerization and lipid binding to catalyze membrane fission in bacteria

Little is known about mechanisms of membrane fission in bacteria despite their requirement for cytokinesis. The only known dedicated membrane fission machinery in bacteria, fission protein B (FisB), is expressed during sporulation in Bacillus subtilis and is required to release the developing spore into the mother cell cytoplasm. Here, we characterized the requirements for FisB-mediated membrane fission. FisB forms mobile clusters of approximately 12 molecules that give way to an immobile cluster at the engulfment pole containing approximately 40 proteins at the time of membrane fission. Analysis of FisB mutants revealed that binding to acidic lipids and homo-oligomerization are both critical for targeting FisB to the engulfment pole and membrane fission. Experiments using artificial membranes and filamentous cells suggest that FisB does not have an intrinsic ability to sense or induce membrane curvature but can bridge membranes. Finally, modeling suggests that homo-oligomerization and trans-interactions with membranes are sufficient to explain FisB accumulation at the membrane neck that connects the engulfment membrane to the rest of the mother cell membrane during late stages of engulfment. Together, our results show that FisB is a robust and unusual membrane fission protein that relies on homo-oligomerization, lipid binding, and the unique membrane topology generated during engulfment for localization and membrane scission, but surprisingly, not on lipid microdomains, negative-curvature lipids, or curvature sensing.

Little is known about how membrane fission occurs in bacteria; this study suggests that the membrane fission protein FisB exploits the unique cellular geometry encountered during sporulation to enable its localization to the fission site through a novel mechanism, where it catalyzes membrane scission.     

Tumor protein D54 binds intracellular nanovesicles via an extended amphipathic region

Tumor protein D54 (TPD54) is an abundant cytosolic protein that belongs to the TPD52 family, a family of four proteins (TPD52, 53, 54, and 55) that are overexpressed in several cancer cells. Even though the functions of these proteins remain elusive, recent investigations indicate that TPD54 binds to very small cytosolic vesicles with a diameter of ca. 30 nm, half the size of classical (e.g., COPI and COPII) transport vesicles. Here, we investigated the mechanism of intracellular nanovesicle capture by TPD54. Bioinformatical analysis suggests that TPD54 contains a small coiled-coil followed by four amphipathic helices (AH1-4), which could fold upon binding to lipid membranes. Limited proteolysis, CD spectroscopy, tryptophan fluorescence, and cysteine mutagenesis coupled to covalent binding of a membrane-sensitive probe showed that binding of TPD54 to small liposomes is accompanied by large structural changes in the amphipathic helix region. Furthermore, site-directed mutagenesis indicated that AH2 and AH3 have a predominant role in TPD54 binding to membranes both in cells and using model liposomes. We found that AH3 has the physicochemical features of an amphipathic lipid packing sensor (ALPS) motif, which, in other proteins, enables membrane binding in a curvature-dependent manner. Accordingly, we observed that binding of TPD54 to liposomes is very sensitive to membrane curvature and lipid unsaturation. We conclude that TPD54 recognizes nanovesicles through a combination of ALPS-dependent and ALPS-independent mechanisms.     

Electrostatic free energy and spontaneous curvature of spherical charged layered membrane

A biophysical model for the equilibrium curvature of a composite membrane element is derived taking into account the mechanical bilayer properties and the adjacent charged protein layers. The minimum of the total free energy density with respect to the curvature of such a membrane curved was estimated from the sum of the electrostatic free energy density of the charges of the membrane and the elastic surface energy density due to bending the lipid bilayer membrane. It was shown that the equilibrium curvature, i.e. the spontaneous curvature, of such a charged composite sandwich-like membrane depends inversely on the bending stiffness of the lipid membrane itself and directly on the charge amount inside and outside the membrane to the second power. Furthermore the geometric and electrostatic structure of the protein layers and the physico-chemical environment conditions are involved. Corresponding to the model developed a "standard RBC" membrane element has a negative spontaneous curvature, accounting for a discocyte RBC shape. The shape change from a discocyte to a more stomatocytic shape (increase in the negative spontaneous curvature) after reducing the charges in the glycocalyx is also explained within this model.     

High cationic charge and bilayer interface-binding helices in a regulatory lipid glycosyltransferase

In the prokaryote Acholeplasma laidlawii, membrane bilayer properties are sensed and regulated by two interface glycosyltransferases (GTs), synthesizing major nonbilayer- (alMGS GT) and bilayer-prone glucolipids. These enzymes are of similar structure, as many soluble GTs, but are sensitive to lipid charge and curvature stress properties. Multivariate and bioinformatic sequence analyses show that such interface enzymes, in relation to soluble ones of similar fold, are characterized by high cationic charge, certain distances between small and cationic amino acids, and by amphipathic helices. Varying surface contents of Lys/Arg pairs and Trp indicate different membrane-binding subclasses. A predicted potential (cationic) binding helix from alMGS was structurally verified by solution NMR and CD. The helix conformation was induced by a zwitterionic as well as anionic lipid environment, and the peptide was confined to the bilayer interface. Bilayer affinity of the peptide, analyzed by surface plasmon resonance, was higher than that for soluble membrane-seeking proteins/peptides and rose with anionic lipid content. Interface intercalation was supported by phase equilibria in membrane lipid mixtures, analyzed by 31P NMR and DSC. An analogous, potentially binding helix has a similar location in the structurally determined Escherichia coli cell wall precursor GT MurG. These two helices have little sequence conservation in alMGS and MurG homologues but maintain their amphipathic character. The evolutionary modification of the alMGS binding helix and its location close to the acceptor substrate site imply a functional importance in enzyme catalysis, potentially providing a mechanism by which glycolipid synthesis will be sensitive to membrane surface charge and intrinsic curvature strain.     

Massive Formation of Intracellular Membrane Vesicles in Escherichia coli by a Monotopic Membrane-bound Lipid Glycosyltransferase

The morphology and curvature of biological bilayers are determined by the packing shapes and interactions of their participant molecules. Bacteria, except photosynthetic groups, usually lack intracellular membrane organelles. Strong overexpression in Escherichia coli of a foreign monotopic glycosyltransferase (named monoglycosyldiacylglycerol synthase), synthesizing a nonbilayer-prone glucolipid, induced massive formation of membrane vesicles in the cytoplasm. Vesicle assemblies were visualized in cytoplasmic zones by fluorescence microscopy. These have a very low buoyant density, substantially different from inner membranes, with a lipid content of ≥60% (w/w). Cryo-transmission electron microscopy revealed cells to be filled with membrane vesicles of various sizes and shapes, which when released were mostly spherical (diameter ≈100 nm). The protein repertoire was similar in vesicle and inner membranes and dominated by the glycosyltransferase. Membrane polar lipid composition was similar too, including the foreign glucolipid. A related glycosyltransferase and an inactive monoglycosyldiacylglycerol synthase mutant also yielded membrane vesicles, but without glucolipid synthesis, strongly indicating that vesiculation is induced by the protein itself. The high capacity for membrane vesicle formation seems inherent in the glycosyltransferase structure, and it depends on the following: (i) lateral expansion of the inner monolayer by interface binding of many molecules; (ii) membrane expansion through stimulation of phospholipid synthesis, by electrostatic binding and sequestration of anionic lipids; (iii) bilayer bending by the packing shape of excess nonbilayer-prone phospholipid or glucolipid; and (iv) potentially also the shape or penetration profile of the glycosyltransferase binding surface. These features seem to apply to several other proteins able to achieve an analogous membrane expansion.