After a vesicle and its target membrane have fused, the SNARE complexes must dissociate to make the individual SNARE proteins available for additional fusion events. Because of the stability of SNARE complexes, which are held together by numerous noncovalent intermolecular interactions, their dissociation depends on additional proteins and the input of energy.
The first clue that dissociation of SNARE complexes re quired the assistance of other proteins came from in vitro transport reactions depleted of certain cytosolic proteins. The observed accumulation of vesicles in these reactions indicated that vesicles could form under these conditions, but were unable to fuse with a target membrane. Eventually two proteins, designated NSF and α-SNAP, were found to be required for ongoing vesicle fusion in the in vitro trans port reaction. The function of NSF in vivo can be blocked selectively by N-ethylmaleimide (NEM), a chemical that re acts with an essential –SH group on NSF (hence the name, NEM-sensitive factor).
Yeast mutants have also contributed to our understanding of SNARE function. Among the class C yeast sec mutants are strains that lack functional Sec18 or Sec17, the yeast counterparts of mammalian NSF and α-SNAP, respectively. When these class C mutants are kept at the nonpermissive temperature, they accumulate ER-to-Golgi transport vesicles; when the cells are shifted to the lower, permissive temperature, the accumulated vesicles are able to fuse with the cis-Golgi.
Subsequent to the initial biochemical and genetic studies that identified NSF and α-SNAP, more sophisticated in vitro transport assays were developed. Using these newer assays, researchers have shown that NSF and α-SNAP proteins are not necessary for actual membrane fusion, but rather are re quired for regeneration of free SNARE proteins. NSF, a hexamer of identical subunits, associates with a SNARE complex with the aid of α-SNAP (soluble NSF attachment protein). The bound NSF then hydrolyzes ATP, releasing sufficient energy to dissociate the SNARE complex (see Figure 1a, step 4). Evidently, the defects in vesicle fusion observed in the earlier in vitro fusion assays and in the yeast mutants after a loss of Sec17 or Sec18 were a consequence of free SNARE proteins rapidly becoming sequestered in undissociated SNARE complexes and thus being unavailable to mediate membrane fusion.
Fig1. Model for docking and fusion of transport vesicles with their target membranes. (a) The proteins shown in this example participate in fusion of secretory vesicles with the plasma membrane, but similar proteins mediate all vesicle-fusion events. Step 1: A Rab protein tethered via a lipid anchor to a secretory vesicle binds to an effector protein complex on the plasma membrane, thereby docking the transport vesicle on the appropriate target mem brane. Step 2: A v-SNARE protein (in this case, VAMP) interacts with the cytosolic domains of the cognate t-SNAREs (in this case, syntaxin and SNAP-25). The very stable coiled-coil SNARE complexes that are formed hold the vesicle close to the target membrane. Step 3 : Fusion of the two membranes immediately follows formation of SNARE complexes, but precisely how this occurs is not known. Step 4: Following membrane fusion, NSF, in conjunction with α-SNAP, binds to the SNARE complexes. The NSF-catalyzed hydrolysis of ATP then drives dissociation of the SNARE complexes, freeing the SNARE proteins for another round of vesicle fusion. Also at this time, Rab·GTP is hydrolyzed to Rab·GDP and dissociates from the Rab effector (not shown). (b) The SNARE complex. Numerous noncovalent interactions between four long α helices, two from SNAP-25 and one each from syntaxin and VAMP, stabilize the coiled-coil structure. See J. E. Rothman and T. Söllner, 1997, Science 276:1212, Y. A. Chen and R. H. Scheller, 2001, Nat. Rev. Mol. Cell Biol. 2:98, and W. Weis and R. Scheller, 1998, Nature 395:328. [Part (b) data from I. Fernandez et al., 1998, Cell 94:841-849, PDB ID 1br0, and R. B. Sutton et al., 1998, Nature 395:347-353, PDB ID 1sfc.]
