Identification of Rab6 as an N-ethylmaleimide-sensitive fusion protein-binding protein

In this study we show the interaction of N-ethylmaleimide-sensitive fusion protein (NSF) with a small GT:P-binding protein, Rab6. NSF is an ATPase inv olved in the vesicular transport within eukaryotic cells. Using the yeast t wo-hybrid system, we have isolated new NSF-binding proteins from the rat lu ng cDNA library. One of them was Rab6, which is invoked in the vesicular tr ansport within the Golgi and trans-Golgi network as a Ras-like GTPase, We d emonstrated that the N-terminal domain of NSF interacted with the C-termina l domain of Rab6, and these proteins were co-immunoprecipitated from the ra t brain extract. This interaction was maintained preferentially in the pres ence of hydrolysable ATP. Recombinant NSF-His(6) can also bind to C-termina l Rab6-glutathione S-transferase under the conditions to allow the ATP hydr olysis. Surprisingly, Rab6 stimulates the ATPase activity of NSF by approx. 2-fold as does alpha -soluble NSF attachment protein receptor. Anti-Rab6 p olyclonal antibodies significantly inhibited the Rab6-stimulated ATPase act ivity of NSF. Furthermore, we found that Rab3 and Rab4 can also associate w ith NSF and stimulate its ATPase activity. Taken together, we propose a mod el in which Rab can form an ATP hydrolysis-regulated complex with NSF, and function as a signalling molecule to deliver the signal of vesicle fusion t hrough the interaction with NSF.