Abstract
Assembly of highly curved membrane structures is essential to cellular physiology. The prevailing view has been that proteins with curvature-promoting structural motifs, such as wedge-like amphipathic helices and crescent-shaped BAR domains, are required for bending membranes. Here we report that intrinsically disordered domains of the endocytic adaptor proteins, Epsin1 and AP180 are highly potent drivers of membrane curvature. This result is unexpected since intrinsically disordered domains lack a well-defined three-dimensional structure. However, in vitro measurements of membrane curvature and protein diffusivity demonstrate that the large hydrodynamic radii of these domains generate steric pressure that drives membrane bending. When disordered adaptor domains are expressed as transmembrane cargo in mammalian cells, they are excluded from clathrin-coated pits. We propose that a balance of steric pressure on the two surfaces of the membrane drives this exclusion. These results provide quantitative evidence for the influence of steric pressure on the content and assembly of curved cellular membrane structures.
Highlights
Of highly curved membrane structures is essential to cellular physiology
To determine whether intrinsically disordered domains could produce highly curved membrane structures, we incubated histagged proteins, including Epsin[1] full-length (FL), Epsin[1] C-terminal domains (CTDs) and AP180 CTD with 200 nm diameter small unilamellar vesicles (SUVs) (Fig. 1b) containing DOGS-Ni-NTA (1,2-dioleoyl-snglycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl]) lipids, which bind with high affinity to histidine residues
Epsin[1] FL, Epsin[1] CTD and AP180 CTD were all able to generate highly curved membrane tubules when exposed to SUVs containing the same concentration of DOGS-Ni-NTA lipids, 20 mol% (Fig. 1d–g; Supplementary Fig. 1)
Summary
Of highly curved membrane structures is essential to cellular physiology. The prevailing view has been that proteins with curvature-promoting structural motifs, such as wedge-like amphipathic helices and crescent-shaped BAR domains, are required for bending membranes. If this pressure is not balanced by crowded molecules on the opposite membrane surface, the resulting imbalance will drive membrane bending towards the more crowded surface This hypothesis, which has been cited to explain membrane bending during coated vesicle assembly[22,23,24,25], amyloid formation[26] and autophagy[27], suggests that proteins with large hydrodynamic radii should make the largest contribution to membrane crowding, since they occupy the most area on the membrane surface[20]. To test the impact of molecular crowding on the assembly of coated vesicles, we examine the ability of clathrincoated pits to internalize cargos that display intrinsically disordered domains on the extracellular plasma membrane surface These experiments provide quantitative evidence that large, crowded cargo molecules exert substantial steric pressure on clathrin-coated pits, markedly decreasing the quantity of cargo molecules that can be encapsulated, in comparison with a small, globular control cargo
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