A conserved membrane attachment site in alpha-SNAP facilitates N-ethylmaleimide-sensitive factor (NSF)-driven SNARE complex disassembly.
The ATPase NSF (N-ethylmaleimide-sensitive factor) and its SNAP (soluble N-ethylmaleimide-sensitive factor attachment protein) cofactor constitute the ubiquitous enzymatic machinery responsible for recycling of the SNARE (SNAP receptor) membrane fusion machinery. The enzyme uses the energy of ATP hydrolysis to dissociate the constituents of the SNARE complex, which is formed during the fusion of a transport vesicle with the acceptor membrane. However, it is still unclear how NSF and the SNAP adaptor work together to take the tight SNARE bundle apart. SNAPs have been reported to attach to membranes independently from SNARE complex binding. We have investigated how efficient the disassembly of soluble and membrane-bound substrates are, comparing the two. We found that SNAPs support disassembly of membrane-bound SNARE complexes much more efficiently. Moreover, we identified a putative, conserved membrane attachment site in an extended loop within the N-terminal domain of alpha-SNAP. Mutation of two highly conserved, exposed phenylalanine residues on the extended loop prevent SNAPs from facilitating disassembly of membrane-bound SNARE complexes. This implies that the disassembly machinery is adapted to attack membrane-bound SNARE complexes, probably in their relaxed cis-configuration.
Winter
Ulrike
U
From the Research Group Structural Biochemistry, Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.
Chen
Xiong
X
Fasshauer
Dirk
D
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism*; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer; D008081:Liposomes; D008561:Membrane Fusion; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D052066:N-Ethylmaleimide-Sensitive Proteins / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D050600:SNARE Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins / Q000235:genetics / Q000378:metabolism*
D008081:Liposomes; D011994:Recombinant Proteins; D050600:SNARE Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D052066:N-Ethylmaleimide-Sensitive Proteins