Quantitative analysis of the interactions between prenyl Rab9, GDP dissociation inhibitor-alpha, and guanine nucleotides.
Rab9 is a Ras-like GTPase required for the transport of mannose 6-phosphate receptors between late endosomes and the trans Golgi network. Rab9 occurs in the cytosol as a complex with GDP dissociation inhibitor (GDI), which we have shown delivers prenyl Rab9 to late endosomes in a functional form. We report here basal rate constants for guanine nucleotide dissociation and GTP hydrolysis for prenyl Rab9. Both rate constants were influenced in part by the hydrophobic environment of the prenyl group. Guanine nucleotide dissociation and GTP hydrolysis rates were lower in the presence of lipid; detergent stimulated intrinsic nucleotide exchange. GDI-alpha inhibited GDP dissociation from prenyl Rab9 by 2.4-fold. GDI-alpha associated with prenyl Rab9 with a KD of 60 nM in 0.1% Lubrol and 23 nM in 0.02% Lubrol. In 0.1% Lubrol, GDI-alpha inhibited GDP dissociation half maximally at 72 +/- 18 nM, consistent with the KD determinations. These data suggest that GDI-alpha associates with prenyl Rab9 with a KD of < or = 23 nM under physiological conditions. Finally, a previously uncharacterized minor form of GDI-alpha inhibited GDP dissociation from prenyl Rab9 by 1.9-fold and bound prenyl Rab9 with a KD of 67 nM in 0.1% Lubrol.
Shapiro
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AD
Department of Biochemistry, Stanford University School of Medicine, California 94305-5307, USA.
Pfeffer
S R
SR
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D020730:Guanine Nucleotide Dissociation Inhibitors; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007700:Kinetics; D011485:Protein Binding; D017368:Protein Prenylation; D018411:Spodoptera; D014162:Transfection; D020691:rab GTP-Binding Proteins
C086796:GDP dissociation inhibitor 1; D020730:Guanine Nucleotide Dissociation Inhibitors; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; C079856:RAB9A protein, human; D020691:rab GTP-Binding Proteins