A POSSIBLE PREDOCKING ATTACHMENT SITE FOR N-ETHYLMALEIMIDE-SENSITIVE FUSION PROTEIN - INSIGHTS FROM IN-VITRO ENDOSOME FUSION

N-Ethylmaleimide-sensitive fusion protein (NSF) is an ubiquitous prote in required for multiple vesicular transport events, We have investiga ted the role of the two nucleotide-binding regions of NSF in endosomal fusion by analyzing NSF mutants in a cell-free system, Our results in dicate that mutations on the first ATP-binding domain, that render a p rotein defective in either ATP binding or ATP hydrolysis, results in a lmost complete inhibition of endosomal fusion, A mutation in the secon d ATP-binding site of NSF was only slightly inhibitory. The inhibitory effect was observed only when the mutant proteins were added at early times during the fusion reaction indicating that NSF may be required for an early step during the docking/fusion process, Binding studies u sing Western blotting reveal that the binding of NSF mutants to endoso mal membranes is differentially affected by Ca2+, Our results indicate that NSF, depending on its nucleotide state, may interact with membra nes via an alternate mechanism, Our findings suggest the existence of a predocking binding site either independent of the docking complex or a site that leads to the formation of the SNAP-SNARE complex (e,g, 20 S particle).