SummaryShallow hydrophobic insertions and crescent-shaped BAR scaffolds promote membrane curvature. Here, we investigate membrane fission by shallow hydrophobic insertions quantitatively and mechanistically. We provide evidence that membrane insertion of the ENTH domain of epsin leads to liposome vesiculation, and that epsin is required for clathrin-coated vesicle budding in cells. We also show that BAR-domain scaffolds from endophilin, amphiphysin, GRAF, and β2-centaurin limit membrane fission driven by hydrophobic insertions. A quantitative assay for vesiculation reveals an antagonistic relationship between amphipathic helices and scaffolds of N-BAR domains in fission. The extent of vesiculation by these proteins and vesicle size depend on the number and length of amphipathic helices per BAR domain, in accord with theoretical considerations. This fission mechanism gives a new framework for understanding membrane scission in the absence of mechanoenzymes such as dynamin and suggests how Arf and Sar proteins work in vesicle scission.

Clathrin-mediated endocytosis (CME) is vital for the internalization of most cell-surface proteins. In CME, plasma membrane-binding clathrin adaptors recruit and polymerize clathrin to form clathrin-coated 'pits' into which cargo is sorted. AP2 is the most abundant adaptor, and is pivotal to CME. By determining a new structure of AP2 that includes the clathrin-binding β2-hinge and developing an AP2-dependent budding assay, we reveal the existence of an autoinhibitory mechanism that prevents clathrin recruitment by cytosolic AP2. A large-scale conformational change driven by the plasma membrane phosphoinositide PtdIns(4,5)P 2 and cargo relieves this autoinhibition, so triggering clathrin recruitment and hence clathrin-coated bud formation. This molecular switching mechanism constitutes an unsuspected layer of regulation that couples AP2's membrane recruitment to its key functions of cargo and clathrin binding.Clathrin adaptors provide an essential physical bridge connecting clathrin, which itself lacks membrane binding activity (1), to the membrane and to embedded transmembrane protein cargo. A central player in CME is the AP2 (Assembly Polypeptide 2) complex, (Figs 1A, S1), which both coordinates CCP formation and binds the many cargo proteins that contain 'acidic dileucine' and Yxxφ endocytic motifs (φ denotes a bulky hydrophobic residue) through its membrane proximal core (2, 3). Cargo binding is activated by a large-scale conformational change from the 'locked' or 'inactive' cytosolic form to an 'open' or 'active' form driven by localization to membranes containing the plasma membrane phosphoinositide PtdIns(4,5)P 2 (4, 5). The C-terminal 'appendages' of the α and β2 subunits bind other clathrin adaptors as well as CCV (clathrin-coated vesicle) assembly and disassembly accessory factors (3,(6)(7)(8) Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts from the β2-trunk binds the N-terminal beta-propeller of the clathrin heavy chain using a canonical clathrin box motif (LLNLD; Fig 1A,B (9)). The β2 appendage domain also binds clathrin, albeit weakly, but both interactions are necessary for robust clathrin binding (10).A version of AP2 comprising full-length β2, μ2 and σ2 subunits, and the α-trunk domain, (FLβ.AP2) (Fig 1B)(11) was expressed in E.coli, avoiding contamination with other CCV components inherent to purification from brain tissue (12, 13). Despite most FLβ.AP2 possessing an intact β2 subunit (Fig 1C-E), it bound clathrin very poorly in pulldowns when immobilized on either glutathione sepharose beads ( Fig 1C) or via its N-terminal His6 tag (similarly positioned to the β2 PtdIns(4,5)P 2 binding site Fig1B (4, 5).) to liposomes containing the nickel-attached lipid NiNTA-DGS ( Fig 1E): in both cases the FLβ.AP2 will be in its locked cytosolic conformation (4). FLβ.AP2 also failed to stimulate clathrin cage assembly efficiently at physiological pH ( Fig 1D). In contrast, the isolated β2 hingeappendage ('GST-β2-h+app', Fig S1) bound clathrin efficiently ( Fig 1C) and stimu...

Membranes of intracellular organelles are characterized by large curvatures with radii of the order of 10–30nm. While, generally, membrane curvature can be a consequence of any asymmetry between the membrane monolayers, generation of large curvatures requires the action of mechanisms based on specialized proteins. Here we discuss the three most relevant classes of such mechanisms with emphasis on the physical requirements for proteins to be effective in generation of membrane curvature. We provide new quantitative estimates of membrane bending by shallow hydrophobic insertions and compare the efficiency of the insertion mechanism with those of the protein scaffolding and crowding mechanisms.

Growing knowledge of the key molecular components involved in biological processes such as endocytosis, exocytosis, and motility has enabled direct testing of proposed mechanistic models by reconstitution. However, current techniques for building increasingly complex cellular structures and functions from purified components are limited in their ability to create conditions that emulate the physical and biochemical constraints of real cells. Here we present an integrated method for forming giant unilamellar vesicles with simultaneous control over (i) lipid composition and asymmetry, (ii) oriented membrane protein incorporation, and (iii) internal contents. As an application of this method, we constructed a synthetic system in which membrane proteins were delivered to the outside of giant vesicles, mimicking aspects of exocytosis. Using confocal fluorescence microscopy, we visualized small encapsulated vesicles docking and mixing membrane components with the giant vesicle membrane, resulting in exposure of previously encapsulated membrane proteins to the external environment. This method for creating giant vesicles can be used to test models of biological processes that depend on confined volume and complex membrane composition, and it may be useful in constructing functional systems for therapeutic and biomaterials applications.lipid bilayer | transmembrane protein | SNARE | microfluidic jetting | synthetic biology