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-His(6) 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 alpha-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.
Han
S Y
SY
School of Biological Sciences, Seoul National University, Seoul 151-742, Korea.
Park
D Y
DY
Park
S D
SD
Hong
S H
SH
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology*; D015723:Gene Library; D005982:Glutathione Transferase / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D008168:Lung / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013997:Time Factors; D020798:Two-Hybrid System Techniques; D001616:beta-Galactosidase / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology*; D020692:rab3 GTP-Binding Proteins / Q000378:metabolism; D020695:rab4 GTP-Binding Proteins / Q000378:metabolism
D018076:DNA, Complementary; C094135:Rab6 protein; D011993:Recombinant Fusion Proteins; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D005982:Glutathione Transferase; D001616:beta-Galactosidase; D020691:rab GTP-Binding Proteins; D020692:rab3 GTP-Binding Proteins; D020695:rab4 GTP-Binding Proteins; D005033:Ethylmaleimide