Cytosolic ATPases, p97 and NSF, are sufficient to mediate rapid membrane fusion

Much recent work has focussed on the role of membrane-bound components in f usion. We show here that p97 and NSF are sufficient to mediate rapid membra ne fusion, Fractionation of cytosol revealed that p97 and its co-factor, p4 7, constitutes the major fusion activity. This was confirmed by depleting p 97 from the cytosol, which resulted in an 80% decrease in fusion, Using pur ified protein, p97 or NSF was found to be sufficient to mediate rapid fusio n in an ATP-dependent manner. A regulatory role was observed for their corr esponding co-factors, p47 and alpha-SNAP, When present at a molar ratio hal f of that of the ATPase, both co-factors increased fusion activity signific antly. Intriguingly, at this ratio the ATPase activity of the complex measu red in solution was at its lowest, suggesting that the co-factor stabilizes the ATP state. The fusion event involved mixing of both leaflets of the op posing membranes and contents of liposomes, We conclude from these data tha t p97, NSF and perhaps other related ATPases catalyse rapid and complete fu sion between lipid bilayers on opposing membranes. This highlights a new ro le for p97 and NSF and prompts a re-evaluation of current fusion models.