A novel Rab9 effector required for endosome-to-TGN transport

J Cell Biol. 1997 Jul 28;138(2):283-90. doi: 10.1083/jcb.138.2.283.

Abstract

Rab9 GTPase is required for the transport of mannose 6-phosphate receptors from endosomes to the trans-Golgi network in living cells, and in an in vitro system that reconstitutes this process. We have used the yeast two-hybrid system to identify proteins that interact preferentially with the active form of Rab9. We report here the discovery of a 40-kD protein (p40) that binds Rab9-GTP with roughly fourfold preference to Rab9-GDP. p40 does not interact with Rab7 or K-Ras; it also fails to bind Rab9 when it is bound to GDI. The protein is found in cytosol, yet a significant fraction (approximately 30%) is associated with cellular membranes. Upon sucrose density gradient flotation, membrane- associated p40 cofractionates with endosomes containing mannose 6-phosphate receptors and the Rab9 GTPase. p40 is a very potent transport factor in that the pure, recombinant protein can stimulate, significantly, an in vitro transport assay that measures transport of mannose 6-phosphate receptors from endosomes to the trans-Golgi network. The functional importance of p40 is confirmed by the finding that anti-p40 antibodies inhibit in vitro transport. Finally, p40 shows synergy with Rab9 in terms of its ability to stimulate mannose 6-phosphate receptor transport. These data are consistent with a model in which p40 and Rab9 act together to drive the process of transport vesicle docking.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Biological Transport
  • Carrier Proteins / chemistry
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism*
  • Cell Membrane / metabolism
  • Cloning, Molecular
  • Endosomes / metabolism*
  • GTP Phosphohydrolases / metabolism*
  • Golgi Apparatus / metabolism*
  • Guanosine Diphosphate / metabolism
  • Guanosine Triphosphate / metabolism
  • Humans
  • Jurkat Cells
  • Molecular Sequence Data
  • Protein Binding
  • Protein Structure, Tertiary
  • Receptor, IGF Type 2 / metabolism
  • Sequence Analysis, DNA
  • Sequence Homology, Amino Acid
  • rab GTP-Binding Proteins*

Substances

  • Carrier Proteins
  • Rab9-binding protein, 40-kDa
  • Receptor, IGF Type 2
  • Guanosine Diphosphate
  • Guanosine Triphosphate
  • GTP Phosphohydrolases
  • RAB9A protein, human
  • rab GTP-Binding Proteins

Associated data

  • GENBANK/Z97074