Biochemical analysis of rab9, a ras-like GTPase involved in protein transport from late endosomes to the trans Golgi network.
rab9 is a ras-like GTPase which has been implicated in the transport of mannose 6-phosphate receptors between late endosomes and the trans Golgi network. We have expressed recombinant rab9 in Escherichia coli, purified the protein to homogeneity, and initiated a biochemical analysis of this enzyme. rab9 hydrolyzed GTP with a rate constant of 0.0052 min-1 at 37 degrees C. rab7, a highly homologous endosomal GTPase, hydrolyzed GTP with a rate constant of 0.0023 min-1 at 37 degrees C. At this temperature, GDP and GTP each dissociated from rab9 with first-order rate constants of 0.017 min-1. GDP and GTP dissociated from rab7 at 37 degrees C with first-order rate constants of 0.0054 and 0.0024 min-1, respectively. We modified the procedure of John et al. (John, J., Sohmen, R., Feuerstein, J., Linke, R., Wittinghofer, A., and Goody, R. (1990) Biochemistry 29, 6058-6065) for the preparation of nucleotide-free ras such that the procedure can now be applied to 1000-fold smaller quantities of protein. Using this method, we prepared microgram quantities of nucleotide-free rab9 in a form which is heat-stable, free of exogenous nucleotide-degrading enzymes and which can be stored at -80 degrees C. At 37 degrees C for GDP and GTP, the second-order rate constants for association with nucleotide-free rab9 were 1.7 x 10(6) M-1 s-1 and 1.2 x 10(5) M-1 s-1, respectively, and equilibrium binding constants were 170 pM and 2.4 nM, respectively.
Shapiro
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AD
Department of Biochemistry, Stanford University School of Medicine, California 94305-5307.
Riederer
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MA
Pfeffer
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SR
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D002845:Chromatography; D003001:Cloning, Molecular; D004926:Escherichia coli; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D009711:Nucleotides / Q000378:metabolism; D015689:Oncogene Protein p21(ras) / Q000378:metabolism; D015388:Organelles / Q000378:metabolism; D020691:rab GTP-Binding Proteins
D001426:Bacterial Proteins; D009711:Nucleotides; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; C079856:RAB9A protein, human; D015689:Oncogene Protein p21(ras); D020691:rab GTP-Binding Proteins