Binding of an N-ethylmaleimide-sensitive fusion protein to Golgi membranes requires both a soluble protein(s) and an integral membrane receptor.

Abstract

Abstract. An N-ethylmaleimide (NEM)-sensitive fusion protein (NSF) has recently been purified on the basis of its ability to restore transport to NEM-inactivated Golgi membranes in a cell-free transport system. NSF is a peripheral membrane protein required for the fusion of transport vesicles. We now report the existence of two novel components that together bind NSF to Golgi membranes in a saturable manner. These components were detected by examining the requirements for reassociation of purified NSF with Golgi membranes in vitro. One component is an integral membrane receptor that is heat sensitive, but resistant to Na2CO3 extraction and to all proteases tested. The second component is a cytosolic factor that is sensitive to both proteases and heat. This soluble NSF attachment protein (SNAP) is largely resistant to NEM and is further distinguished from NSF by chromatography. SNAP appears to act stoichiometrically in promoting a high-affinity interaction between NSF and the membrane receptor. Because NSF promotes vesicle fusion, it seems likely that these two new factors that allow NSF to bind to the membrane are also part of the fusion machinery.

Cryo-EM structure of SNAP-SNARE assembly in 20S particle

“…Most α helices of α-SNAP are clearly visible in the map except for the very C-terminal end, near which there are weak densities possibly corresponding to the N-domain of NSF known to interact with α-SNAP's C terminus [19][20][21][22][23]. The four-helical bundle of the SNARE complex fits perfectly in the central left-handed four threadlike densities albeit with the side chain of each residue in the helix unsolved.…”

“…We also showed that the N-domains of NSFs move to the periphery of the complex when ATP is hydrolyzed to ADP. As the C-terminal domain of α-SNAP interacts directly with the N-domain of NSF [19][20][21][22][23], the movement of the N-domain in the 20S particle very likely causes the similar movement of α-SNAP. It is therefore plausible that, within an assembled 20S particle, a conformational change of the NSF triggered by ATP hydrolysis would be transduced to the tetrameric α-SNAP for it to unwind the SNARE complex (Figure 4).…”

“…D2 domain is responsible for maintaining NSF as a hexamer, while D1 domain is catalytically active and functions to hydrolyze ATP accompanied by a major conformational change of itself as well as the N-domain attached to it [16][17][18]. Previous studies indicated that the N-domain of NSF interacts with the C-terminal end of α-SNAP, which mediates the interaction of NSF and the SNARE complex [19][20][21][22][23]. Crystallographic study has revealed the structure of Sec17, a yeast homolog of α-SNAP, composed of an N-terminal twisted sheet of α-helical hairpins and a C-terminal α-helical bundle [24].…”

Cytosolic ARFs are required for vesicle formation but not for cell-free intra-Golgi transport: evidence for coated vesicle-independent transport.

“…These include a 115 kDa peripheral membrane protein (Waters et al, 1992), an N-ethylmaleimide sensitive fusion protein (NSF; Block et al, 1988) and soluble NSF-attachment proteins, (SNAPS; Clary et al, 1990) which recruit NSF to specific receptors on Golgi membranes (Weidman et al, 1989). Recent studies have established that short peptides derived from the N-terminus of ARFs block the intra-Golgi transport assay .…”

Le principe de la fusion membranaire dans la cellule

“…Il paraissait vraisemblable que les SNARE seraient insérées directement dans les membranes parce que les récepteurs de SNAP gardent la capacité de lier SNAP, même après extraction des membranesà l'aide d'un traitement alcalin fort, dont la brutalité enlève tout ce qui n'est pas protéines membranaires intégrales (Weidman et al, 1989). La purification des protéines de membrane devint notre priorité absolue, car nous supposions que la fusion des bicouches lipidiques nécessiterait des protéines ancréesà la membrane.…”