Identification of Rab6 as an N-ethylmaleimide-sensitive fusion protein-binding protein
Copyright policy )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 GTP-binding protein, Rab6. NSF is an ATPase involved in the vesicular transport within eukaryotic cells. Using the yeast two-hybrid system, we have isolated new NSF-binding proteins from the rat lung cDNA library. One of them was Rab6, which is involved in the vesicular transport within the Golgi and trans-Golgi network as a Ras-like GTPase. We demonstrated that the N-terminal domain of NSF interacted with the C-terminal domain of Rab6, and these proteins were co-immunoprecipitated from the rat brain extract. This interaction was maintained preferentially in the presence of hydrolysable ATP. Recombinant NSF-His6 can also bind to C-terminal Rab6–glutathione S-transferase under the conditions to allow the ATP hydrolysis. Surprisingly, Rab6 stimulates the ATPase activity of NSF by approx. 2-fold as does α-soluble NSF attachment protein receptor. Anti-Rab6 polyclonal antibodies significantly inhibited the Rab6-stimulated ATPase activity of NSF. Furthermore, we found that Rab3 and Rab4 can also associate with NSF and stimulate its ATPase activity. Taken together, we propose a model 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 through the interaction with NSF.
- Seoul National University Korea (Republic of)
DNA, Complementary, Hydrolysis, Blotting, Western, Molecular Sequence Data, Golgi Apparatus, Models, Biological, Precipitin Tests, Mice, Adenosine Triphosphate, Microscopy, Fluorescence, Ethylmaleimide, Cricetinae, Animals, Humans, Amino Acid Sequence, Guanosine Triphosphate, Lung, Gene Library, Glutathione Transferase, Protein Binding
DNA, Complementary, Hydrolysis, Blotting, Western, Molecular Sequence Data, Golgi Apparatus, Models, Biological, Precipitin Tests, Mice, Adenosine Triphosphate, Microscopy, Fluorescence, Ethylmaleimide, Cricetinae, Animals, Humans, Amino Acid Sequence, Guanosine Triphosphate, Lung, Gene Library, Glutathione Transferase, Protein Binding
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