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-phos phate receptors between late endosomes and the trans Golgi network. Ra b9 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 nucl eotide dissociation and GTP hydrolysis for prenyl Rab9. Both rate cons tants were influenced in part by the hydrophobic environment of the pr enyl group. Guanine nucleotide dissociation and GTP hydrolysis rates w ere lower in the presence of lipid; detergent stimulated intrinsic nuc leotide exchange. GDI-alpha inhibited GDP dissociation from prenyl Rab 9 by 2.4-fold. GDI-alpha associated with prenyl Rab9 with a K-D 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 K-D determinations. These data suggest that GDI-alpha associ ates with prenyl Rab9 with a K-D of less than or equal to 23 nM under physiological conditions. Finally, a previously uncharacterized minor form of GDI-alpha inhibited GDP dissociation from prenyl Babe by 1.9-f old and bound prenyl Rab9 with a K-D of 67 nM in 0.1% Lubrol.