Unraveling the mechanism of the vesicle transport ATPase NSF, the N-ethylmaleimide-sensitive factor.

The transport of cargo in eukaryotic cells is mediated by the movement of membranous vesicles that pinch off from one membrane and fuse with another. An essential part of this process is the interaction between SNARE (soluble NSF attachment protein receptors) proteins from the vesicle (v-SNARE) and target (t-SNARE) membranes. The resulting SNARE complexes are parallel fourhelix coiled-coil structures with melting temperatures between 70 and 90 °C, and it is likely that, at least in part, the free energy of SNARE complex formation drives bilayer fusion (1). Regulating the assembly and disassembly of SNARE complexes is thus an important aspect of vesicular transport. The hexameric ATPase N-ethylmaleimide-sensitive factor (NSF) uses energy from ATP hydrolysis to dissociate SNARE complexes after membrane fusion, allowing the individual SNARE proteins to be recycled for subsequent rounds of fusion (1). NSF binds to and dissociates SNARE complexes only in the presence of the adaptor protein, a-SNAP (soluble NSF attachment protein). a-SNAP interacts directly with the SNARE complex and with ATP-bound NSF to form the so-called “20 S particle” (2, 3). In the 20 S particle, a-SNAP stimulates the ATPase activity of NSF, leading to SNARE complex disassembly (Fig. 1) (4, 5). Specific vand t-SNAREs are associated with each intercompartmental transport step, but NSF and a-SNAP are general cytosolic factors that can disassemble the SNARE complexes from most, if not all, intracellular transport steps (1). The NSF protomer contains three domains: an N-terminal domain, NSF-N (residues 1–205), responsible for interaction with the a-SNAP-SNARE complex and two homologous ATP-binding domains, NSF-D1 (residues 206–488) and NSF-D2 (residues 489– 744) (3). NSF-D1 is an active ATPase that provides the driving force for SNARE complex disassembly (6, 7). NSF-D1 must bind ATP to interact with the a-SNAP-SNARE complex. NSF-D2 is responsible for maintaining NSF as a hexamer (6). It has higher affinity for ATP than NSF-D1 (8) but has no significant ATPase activity. Nucleotide binding by NSF-D2 is, however, important for hexamerization. The sequences of NSF-D1 and NSF-D2 place NSF in the AAA (ATPases associated with various cellular activities (9)) superfamily (10). AAA proteins, which contain at least one copy of a conserved ;230-amino acid cassette, are involved in a wide variety of cellular roles, including membrane fusion, proteosome regulation, transcription, organelle biogenesis, and microtubule transport and regulation (10, 11). Despite this functional diversity, the ability to assemble or disassemble multisubunit macromolecular complexes, or to fold or unfold polypeptides, appears to be common to the family. Here, we focus on recent advances that are helping provide a basis for understanding the physical mechanisms that underlie the role of NSF in SNARE complex disruption.