Autophagy, an important catabolic pathway implicated in a broad spectrum of human diseases, begins by forming double membrane autophagosomes that engulf cytosolic cargo and ends by

SummaryEvolutionarily conserved SNARE (Soluble N-ethylmaleimide sensitive factor Attachment protein REceptors) proteins form a complex that drives fusion between membranes in eukaryotes. SNARE complexes are disassembled by the ATPase NSF (N-ethylmaleimide Sensitive Factor), together with SNAP (Soluble NSF Attachment Protein) proteins, making individual SNAREs available for a subsequent round of fusion. Here we report structures of ATP- and ADP-bound NSF, and the NSF/SNAP/SNARE (20S) supercomplex determined by single-particle electron cryomicroscopy at near-atomic to sub-nanometer resolution without imposing symmetry. Large, potentially force-generating, conformational differences exist between ATP- and ADP-bound NSF. The 20S supercomplex exhibits broken symmetry, transitioning from six-fold symmetry of the NSF ATPase domains, to pseudo four-fold symmetry of the SNARE complex. SNAPs are interacting with the SNARE complex with an opposite structural twist, suggesting an unwinding mechanism. The interfaces between NSF, SNAPs, and SNAREs exhibit characteristic electrostatic patterns, suggesting how one NSF/SNAP species can act on many different SNARE complexes.

α-Synuclein is a presynaptic protein that is implicated in Parkinson's and other neurodegenerative diseases. Physiologically, native α-synuclein promotes presynaptic SNARE-complex assembly, but its molecular mechanism of action remains unknown. Here, we found that native α-synuclein promotes clustering of synaptic-vesicle mimics, using a single-vesicle optical microscopy system. This vesicle-clustering activity was observed for both recombinant and native α-synuclein purified from mouse brain. Clustering was dependent on specific interactions of native α-synuclein with both synaptobrevin-2/VAMP2 and anionic lipids. Out of the three familial Parkinson's disease-related point mutants of α-synuclein, only the lipid-binding deficient mutation A30P disrupted clustering, hinting at a possible loss of function phenotype for this mutant. α-Synuclein had little effect on Ca2+-triggered fusion in our reconstituted single-vesicle system, consistent with in vivo data. α-Synuclein may therefore lead to accumulation of synaptic vesicles at the active zone, providing a ‘buffer’ of synaptic vesicles, without affecting neurotransmitter release itself.DOI: http://dx.doi.org/10.7554/eLife.00592.001