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

Biochem J. 2000 Nov 15;352 Pt 1(Pt 1):165-73.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Amino Acid Sequence
  • Animals
  • Blotting, Western
  • Cricetinae
  • DNA, Complementary / metabolism
  • Ethylmaleimide / pharmacology*
  • Gene Library
  • Glutathione Transferase / metabolism
  • Golgi Apparatus / metabolism
  • Guanosine Triphosphate / metabolism
  • Humans
  • Hydrolysis
  • Lung / metabolism
  • Mice
  • Microscopy, Fluorescence
  • Models, Biological
  • Molecular Sequence Data
  • Precipitin Tests
  • Protein Binding
  • Protein Structure, Tertiary
  • Rats
  • Rats, Sprague-Dawley
  • Recombinant Fusion Proteins / metabolism*
  • Sequence Homology, Amino Acid
  • Signal Transduction
  • Time Factors
  • Two-Hybrid System Techniques
  • beta-Galactosidase / metabolism
  • rab GTP-Binding Proteins / chemistry
  • rab GTP-Binding Proteins / metabolism*
  • rab GTP-Binding Proteins / physiology*
  • rab3 GTP-Binding Proteins / metabolism
  • rab4 GTP-Binding Proteins / metabolism

Substances

  • DNA, Complementary
  • Rab6 protein
  • Recombinant Fusion Proteins
  • Guanosine Triphosphate
  • Adenosine Triphosphate
  • Glutathione Transferase
  • beta-Galactosidase
  • rab GTP-Binding Proteins
  • rab3 GTP-Binding Proteins
  • rab4 GTP-Binding Proteins
  • Ethylmaleimide

Associated data

  • GENBANK/AF091834
  • GENBANK/AF148210