Cryoem Studies of Membrane-Protein Interactions

Abstract

The processes of membrane fission and fusion both involve an intermediate state called hemi-fission or hemi-fusion, respectively. This intermediate is fundamental and controls the outcome of both processes. During hemi-fusion/fission, the outer leaflet of a budding vesicle is still connected to the plasma membrane, while the inner leaflet has gone through separation/fission. It is particularly critical for fast-paced endocytosis in neural synapses. At the budding neck, this intermediate assumes the form of a tubular micelle. We investigate the interactions of neural synaptic proteins alpha-synuclein and endophilin with lipid vesicles and their ability of membrane remodeling in vitro by cryo-EM, and found that both proteins are capable of inducing the formation of tubular micelles.Alpha-synuclein, whose amyloidal aggregates are a hallmark of Parkinson's disease, has been suggested to have an interaction with biological membranes that is crucial in synaptic endocytosis. Alpha-synuclein changes the conformation to alpha-helix upon binding to membranes, however, little is known about the molecular mechanisms of this interaction. We studied vesicle tubulation by alpha-synuclein, presumably induced by its amphipathic helix wedging membranes, thereby altering their curvature. We investigated its structural profile and found that alpha-synuclein transforms vesicles into tubular micelles and further into lipid particles, concomitantly with the synuclein conformational change.Endophilin is a member of the BAR superfamily. The current view is that it recognizes a nascent endocytic vesicle protruding from the synapse by its crescent shape. Its binding then alters the membrane curvature, leading to neck formation and the recruitment of dynamin to this site. On exposing vesicles to endophilin, we observed the formation of tubes varying in diameter from 70 A to 400 A, with the thinnest tubes to be a protein-coated tubular micelle. The degree of the squeezing depends on the local density of endophilin on the membrane.