A novel Rab9 effector required for endosome-to-TGN transport.
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.
Díaz
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Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305-5307, USA.
Schimmöller
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F
Pfeffer
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D000595:Amino Acid Sequence; D001483:Base Sequence; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D011992:Endosomes / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017527:Receptor, IGF Type 2 / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D020691:rab GTP-Binding Proteins
D002352:Carrier Proteins; C107297:Rab9-binding protein, 40-kDa; D017527:Receptor, IGF Type 2; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; C079856:RAB9A protein, human; D020691:rab GTP-Binding Proteins