The protein coat in membrane fusion: lessons from fission

Multiple cell biological processes involve two opposite rearrangements of membrane configuration, referred to as fusion and fission. While membrane intermediates in protein-mediated fusion have been studied in some detail, the global force which drives sequential stages of the fusion reaction from early local intermediates to an expanding fusion pore remains unknown. Fusion proceeds via stages, which are analogous but in the opposite direction to that of membrane budding-off and fission driven by protein coats. On the basis of this analogy, we propose that an interconnected coat formed by membrane-bound activated fusion proteins surrounding the membrane contact zone generates the driving force for fusion. This fusion protein coat has a strongly curved intrinsic shape opposite to that of the protein coat driving fission. To relieve internal stresses, the fusion protein coat spontaneously bends out of the initial shape of the membrane surface. This bending produces elastic stresses in the underlying lipid bilayer and drives its fusion with the apposing membrane. The hypothesis that 'bystander' proteins (i.e. fusion proteins outside the contact zone) generate the driving force for fusion offers a new interpretation for a number of known features of the fusion reaction mediated by the prototype fusion protein, influenza hemagglutinin, and might bring new insights into mechanisms of other fusion reactions.     

Curvature-dependent lateral distribution of raft markers in the human erythrocyte membrane

The distribution of raft markers in curved membrane exvaginations and invaginations, induced in human erythrocytes by amphiphile-treatment or increased cytosolic calcium level, was studied by fluorescence microscopy. Cholera toxin subunit B and antibodies were used to detect raft components. Ganglioside GM1 was enriched in membrane exvaginations (spiculae) induced by cytosolic calcium and amphiphiles. Stomatin and the cytosolic proteins synexin and sorcin were enriched in spiculae when induced by cytosolic calcium, but not in spiculae induced by amphiphiles. No enrichment of flotillin-1 was detected in spiculae. Analyses of the relative protein content of released exovesicles were in line with the microscopic observations. In invaginations induced by amphiphiles, the enrichment of ganglioside GM1, but not of the integral membrane proteins flotillin-1 and stomatin, was observed. Based on the experimental results and theoretical considerations we suggest that membrane skeleton-detached, laterally mobile rafts may sort into curved or flat membrane regions dependent on their intrinsic molecular shape and/or direct interactions between the raft elements.     

Role of curvature and phase transition in lipid sorting and fission of membrane tubules

We have recently developed a minimal system for generating long tubular nanostructures that resemble tubes observed in vivo with biological membranes. Here, we studied membrane tube pulling in ternary mixtures of sphingomyelin, phosphatidylcholine and cholesterol. Two salient results emerged: the lipid composition is significantly different in the tubes and in the vesicles; tube fission is observed when phase separation is generated in the tubes. This shows that lipid sorting may depend critically on both membrane curvature and phase separation. Phase separation also appears to be important for membrane fission in tubes pulled out of giant liposomes or purified Golgi membranes.     

ArfGAP1 generates an Arf1 gradient on continuous lipid membranes displaying flat and curved regions

ArfGAP1, which promotes GTP hydrolysis on the small G protein Arf1 on Golgi membranes, interacts preferentially with positively curved membranes through its amphipathic lipid packing sensor (ALPS) motifs. This should influence the distribution of Arf1‐GTP when flat and curved regions coexist on a continuous membrane, notably during COPI vesicle budding. To test this, we pulled tubes from giant vesicles using molecular motors or optical tweezers. Arf1‐GTP distributed on the giant vesicles and on the tubes, whereas ArfGAP1 bound exclusively to the tubes. Decreasing the tube radius revealed a threshold of R≈35 nm for the binding of ArfGAP1 ALPS motifs. Mixing catalytic amounts of ArfGAP1 with Arf1‐GTP induced a smooth Arf1 gradient along the tube. This reflects that Arf1 molecules leaving the tube on GTP hydrolysis are replaced by new Arf1‐GTP molecules diffusing from the giant vesicle. The characteristic length of the gradient is two orders of magnitude larger than a COPI bud, suggesting that Arf1‐GTP diffusion can readily compensate for the localized loss of Arf1 during budding and contribute to the stability of the coat until fission.     

Cryo-EM study of patched in lipid nanodisc suggests a structural basis for its clustering in caveolae

1. Download : Download high-res image (162KB)
2. Download : Download full-size image

     

Curvature-based sorting of eight lipid types in asymmetric buckled plasma membrane models

Curvature is a fundamental property of biological membranes and has essential roles in cellular function. Bending of membranes can be induced by their lipid and protein compositions, as well as peripheral proteins, such as those that make up the cytoskeleton. An important aspect of membrane function is the grouping of lipid species into microdomains, or rafts, which serve as platforms for specific biochemical processes. The fluid mosaic model of membranes has evolved to recognize the importance of curvature and leaflet asymmetry, and there are efforts toward evaluating their functional roles. This work investigates the effect of curvature on the sorting of lipids in buckled asymmetric bilayers containing eight lipid types, approximating an average mammalian plasma membrane, through coarse-grained (CG) molecular dynamics (MD) simulations with the Martini force field. The simulations reveal that 1) leaflet compositional asymmetry can induce curvature asymmetry, 2) lipids are sorted by curvature to different extents, and 3) curvature-based partitioning trends show moderate to strong correlations with lipid molecular volumes and head to tail bead ratios, respectively. The findings provide unique insights into the role of curvature in membrane organization, and the curvature-based sorting trends should be useful references for later investigations and potentially interpreting the functional roles of specific lipids.     

Shaping the flavivirus replication complex: It is curvaceous!

Flavivirus replication is intimately involved with remodelled membrane organelles that are compartmentalised for different functions during their life cycle. Recent advances in lipid analyses and gene depletion have identified a number of host components that enable efficient virus replication in infected cells. Here, we describe the current understanding on the role and contribution of host lipids and membrane bending proteins to flavivirus replication, with a particular focus on the components that bend and shape the membrane bilayer to induce the flavivirus‐induced organelles characteristic of infection.     

Physical factors that affect the number and size of Golgi cisternae

Despite continual membrane reorganization in the Golgi complex, the number of cisternae in a Golgi stack is a stable parameter. The cisternal number is conserved within any given cell line and also after Golgi reassembly, e.g. following brefeldin-A-induced disruption. However, the factors that determine the cisternal number in a single Golgi stack remain to be fully determined. We propose a simple mechanical model of the Golgi stack and present a theoretical analysis of different physical factors that may affect the number of cisternae in a Golgi stack. The model takes into account the Golgi membrane bending elasticity, which is related to the membrane curvature, and the adhesion, which holds the cisternae together. The analysis shows that the equilibrium configuration of the Golgi stack can be regarded as a balance between these two effects - the adhesion, which tends to increase the number of cisternae, is opposed by the membrane resistance to bending, which does not favor highly curved cisternal rims. The adhesion strength that is needed to hold together a typical stack is calculated. In addition, the model is used to analyze changes in the cisternal numbers as a controlled traffic wave enters a Golgi stack and increases the amount of the membrane in that stack.     

Spatial Control of Epsin-induced Clathrin Assembly by Membrane Curvature

Epsins belong to the family of highly conserved clathrin-associated sorting proteins that are indispensable for clathrin-mediated endocytosis, but their precise functions remain unclear. We have developed an assay system of budded supported membrane tubes displaying planar and highly curved membrane surfaces to analyze intrinsic membrane curvature preference shown by clathrin-associated sorting proteins. Using real-time fluorescence microscopy, we find that epsin preferentially partitions to and assembles clathrin on highly curved membrane surfaces. Sorting of epsin to regions of high curvature strictly depends on binding to phosphatidylinositol 4,5-bisphosphate. Fluorescently labeled clathrins rapidly assemble as foci, which in turn cluster epsin, while maintaining tube integrity. Clathrin foci grow in intensity with a typical time constant of ∼75 s, similar to the time scales for coated pit formation seen in cells. Epsin therefore effectively senses membrane curvature to spatially control clathrin assembly. Our results highlight the potential role of membrane curvature in orchestrating the myriad molecular interactions necessary for the success of clathrin-mediated membrane budding.     

Features critical for membrane binding revealed by DivIVA crystal structure

DivIVA is a conserved protein in Gram‐positive bacteria that localizes at the poles and division sites, presumably through direct sensing of membrane curvature. DivIVA functions as a scaffold and is vital for septum site selection during vegetative growth and chromosome anchoring during sporulation. DivIVA deletion causes filamentous growth in Bacillus subtilis, whereas overexpression causes hyphal branching in Streptomyces coelicolor. We have determined the crystal structure of the N‐terminal (Nt) domain of DivIVA, and show that it forms a parallel coiled‐coil. It is capped with two unique crossed and intertwined loops, exposing hydrophobic and positively charged residues that we show here are essential for membrane binding. An intragenic suppressor introducing a positive charge restores membrane binding after mutating the hydrophobic residues. We propose that the hydrophobic residues insert into the membrane and that the positively charged residues bind to the membrane surface. A low‐resolution crystal structure of the C‐terminal (Ct) domain displays a curved tetramer made from two parallel coiled‐coils. The Nt and Ct parts were then merged into a model of the full length, 30 nm long DivIVA protein.     

Curvature factor and membrane solubilization, with particular reference to membrane rafts

The composition of membrane rafts (cholesterol/sphingolipid-rich domains) cannot be fully deduced from the analysis of a detergent-resistant membrane fraction after solubilization in Triton X-100 at 4°C. It is hypothesized that the membrane curvature-dependent lateral distribution of membrane components affects their solubilization. The stomatocytogenic, Triton X-100, cannot effectively solubilize membrane components, especially with regard to the outward membrane curvature.     

Phospholipid‐flipping activity of P4‐ATPase drives membrane curvature

P4‐ATPases are phospholipid flippases that translocate phospholipids from the exoplasmic/luminal to the cytoplasmic leaflet of biological membranes. All P4‐ATPases in yeast and some in other organisms are required for membrane trafficking; therefore, changes in the transbilayer lipid composition induced by flippases are thought to be crucial for membrane deformation. However, it is poorly understood whether the phospholipid‐flipping activity of P4‐ATPases can promote membrane deformation. In this study, we assessed membrane deformation induced by flippase activity via monitoring the extent of membrane tubulation using a system that allows inducible recruitment of Bin/amphiphysin/Rvs (BAR) domains to the plasma membrane (PM). Enhanced phosphatidylcholine‐flippase activity at the PM due to expression of ATP10A, a member of the P4‐ATPase family, promoted membrane tubulation upon recruitment of BAR domains to the PM. This is the important evidence that changes in the transbilayer lipid composition induced by P4‐ATPases can deform biological membranes.     

Membrane Fusion

The fusion of biological membranes results in two bilayer-based membranes merging into a single membrane. In this process the lipids have to undergo considerable rearrangement. The nature of the intermediates that are formed during this rearrangement has been investigated. Certain fusion proteins facilitate this process. In many cases short segments of these fusion proteins have a particularly important role in accelerating the fusion process. Studies of the interaction of model peptides with membranes have allowed for increased understanding at the molecular level of the mechanism of the promotion of membrane fusion by fusion proteins. There is an increased appreciation of the roles of several independent segments of fusion proteins in promoting the fusion process.Many of the studies of the fusion of biological membranes have been done with the fusion of enveloped viruses with other membranes. One reason for this is that the number of proteins involved in viral fusion is relatively simple, often requiring only a single protein. For many enveloped viruses, the structure of their fusion proteins has certain common elements, suggesting that they all promote fusion by an analogous mechanism. Some aspects of this mechanism also appears to be common to intracellular fusion, although several proteins are involved in that process which is more complex and regulated than is fusion.     

Membrane curvature affects the stability and folding kinetics of bacteriorhodopsin

Biological membrane is crucial for the function, stability and folding of membrane proteins. By studying the stability and folding kinetics of bacteriorhodopsin (bR) in lipid vesicles with different sizes, here we report the influence of membrane curvature (vesicle size) on the stability and folding kinetics of bR. The results show that both the stability and folding kinetics of bR can be significantly changed when reconstituted into mimic membranes with different curvatures. The stability of bR decreases and unfolding rate of bR increases with the growth of vesicle size, i.e. decrease of membrane curvature. Our results suggest that it is possible to regulate the properties of membrane proteins by changing the curvature of membranes.     

Lysophospholipids Facilitate COPII Vesicle Formation

1. Download high-res image (222KB)
2. Download full-size image

     

Patching of gangliosideM1 in human erythrocytes – distribution of CD47 and CD59 in patched and curved membrane

Membrane rafts may act as platforms for membrane protein signalling. Rafts have also been implicated in the sorting of membrane components during membrane budding. We have studied by fluorescence microscopy cross-linking of ganglioside GM1 in the human erythrocyte membrane, and how membrane proteins CD47 and CD59 distribute in GM1 patched discoid cells and calcium-induced echinocytic cells. Patching of ganglioside_M1 (GM1) by cholera toxin subunit B (CTB) plus anti-CTB resulted in the formation of usually 40–60 GM1 patches distributed over the membrane in discoid erythrocytes. Pre-treatment of erythrocytes with methyl-β-cyclodextrin abolished GM1 patching. GM1 patching was insensitive to pre-fixation (paraformaldehyde) of cells. Patching of GM1 did not affect the discoid shape of erythrocytes. Membrane proteins CD47 and CD59 did not accumulate into GM1 patches. No capping of patches occurred. GM1 accumulated in calcium-induced echinocytic spiculae. Also CD59, but not CD47, accumulated in spiculae. However, CD59 showed a low degree of co-localization with GM1 and frequently accumulated in different spiculae than GM1. In conclusion, our study describes a novel method for examining properties and composition of rafts. The study characterizes raft patching in the human erythrocyte membrane and emphasizes the mobility and ‘echinophilicity’ of GM1. Glycosyl phosphatidylinositol-anchored CD59 was identified as a mobile ‘echinophilic’ but ‘raftophobic_GM1’ protein. Largely immobile CD47 showed no segregation.     

The Cytoplasmic Domain of the SARS-CoV-2 Envelope Protein Assembles into a β-Sheet Bundle in Lipid Bilayers

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) envelope (E) protein forms a pentameric ion channel in the lipid membrane of the endoplasmic reticulum Golgi intermediate compartment (ERGIC) of the infected cell. The cytoplasmic domain of E interacts with host proteins to cause virus pathogenicity and may also mediate virus assembly and budding. To understand the structural basis of these functions, here we investigate the conformation and dynamics of an E protein construct (residues 8–65) that encompasses the transmembrane domain and the majority of the cytoplasmic domain using solid-state NMR. ¹³C and ¹⁵N chemical shifts indicate that the cytoplasmic domain adopts a β-sheet-rich conformation that contains three β-strands separated by turns. The five subunits associate into an umbrella-shaped bundle that is attached to the transmembrane helices by a disordered loop. Water-edited NMR spectra indicate that the third β-strand at the C terminus of the protein is well hydrated, indicating that it is at the surface of the β-bundle. The structure of the cytoplasmic domain cannot be uniquely determined from the inter-residue correlations obtained here due to ambiguities in distinguishing intermolecular and intramolecular contacts for a compact pentameric assembly of this small domain. Instead, we present four structural topologies that are consistent with the measured inter-residue contacts. These data indicate that the cytoplasmic domain of the SARS-CoV-2 E protein has a strong propensity to adopt β-sheet conformations when the protein is present at high concentrations in lipid bilayers. The equilibrium between the β-strand conformation and the previously reported α-helical conformation may underlie the multiple functions of E in the host cell and in the virion.