A Conserved Membrane Attachment Site in α-SNAP Facilitates N-Ethylmaleimide-sensitive Factor (NSF)-driven SNARE Complex Disassembly

Abstract

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 ␣-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.All intracellular vesicle transport processes, ranging from secretion in yeast to neurotransmitter release in the brain, depend on the ability of membranes to fuse with each other. Intracellular fusion is mediated by the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) 3 proteins on opposing membranes, which assemble in transconfiguration into four-helix bundle complexes, bringing the membranes into close proximity (for an overview see Refs.

Three αSNAP and 10 ATP Molecules Are Used in SNARE Complex Disassembly by N-ethylmaleimide-sensitive Factor (NSF)

“…We found, as has been noted by others (32), that the presence of Cl Ϫ diminishes NSF activity. Although NSF activity can be measured under very low ionic strength conditions to circumvent this problem (32), we found that a buffer containing carboxylates as the predominant anions, which more closely mimics physiological conditions, supports NSF activity (23). Disassembly of the SC in this buffer was measured by the decrease in anisotropy of labeled VAMP2 (VAMP2-A488) under steady-state conditions (Fig.…”

“…Our design of the trimerized ␣SNAP 3 was inspired by the observation that a 20-fold lower ␣SNAP concentration was needed to support the NSF-mediated disassembly of liposomeanchored SNARE complexes versus soluble complexes, such as those used in this paper (23). In that work, it was suggested that the enhanced effect of lipid-associated ␣SNAP arises from an increase in local concentration due to restriction to the twodimensional bilayer, or perhaps that the lipid association produces a conformational change in ␣SNAP.…”

“…The GST tag was removed on column using thrombin (Sigma-Aldrich), and the cleaved protein was purified by size exclusion chromatography (S200 26/60) and anion exchange (MonoQ) (23). Fractions containing pure ␣SNAP were pooled and concentrated using Ultracel 10K centrifugal filters.…”

“…In qualitative pull-down assays, a trimeric ␣SNAP produced by adding an N-terminal coiled-coil domain was shown to bind NSF in the absence of SNARE complex, whereas monomeric ␣SNAP bound NSF only in the presence of the SNARE complex (19), but the effect of trimerization on NSF ATPase or SNARE complex disassembly activities was not tested. The NSF N-domain interacts with the C-terminal domain of ␣SNAP (19,22), and ␣SNAP contains a membrane-associated loop near its N terminus that probably orients the C terminus of ␣SNAP toward the cytosol (23) (Fig. 1A).…”

The Intertransmembrane Region of Kaposi's Sarcoma-Associated Herpesvirus Modulator of Immune Recognition 2 Contributes to B7-2 Downregulation

“…4). Since aromatic amino acids, including phenylalanine, are frequently utilized for membrane anchoring (24,34,37,38,41), the hydrophobicity of the six-member ring structure is assumed to be critical for ITM-mediated B7-2 downregulation. This hypothesis was supported by site saturation mutagenesis of Phe119; tyrosine, which also contains a six-member ring structure, could function instead of phenylalanine at position 119.…”