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Rab5-dependent endosome fusion is sensitive to the phosphoinositide 3-kinase inhibitor, wortmannin. It has been proposed that phosphoinositide 3-kinase activity may be required for activation of rab5 by influencing its nucleotide cycle such as to promote its active GTP state. In this report we demonstrate that endosome fusion remains sensitive to wortmannin despite preloading of endosomes with stimulatory levels of a GTPase-defective mutant rab5(Q79L) or of a xanthosine triphosphate-binding mutant, rab5(D136N), in the presence of the nonhydrolysable analogue XTPgammaS. These results suggest that activation of rab5 cannot be the principal function of the wortmannin-sensitive factor on the endosome fusion pathway. This result is extrapolated to all GTPases by demonstrating that endosome fusion remains wortmannin sensitive despite prior incubation with the nonhydrolysable nucleotide analogue GTPgammaS. Consistent with these results, direct measurement of clathrin-coated vesicle-stimulated nucleotide dissociation from exogenous rab5 was insensitive to the presence of wortmannin. A large excess of rab5(Q79L), beyond levels required for maximal stimulation of the fusion assay, afforded protection against wortmannin inhibition, and partial protection was also observed with an excess of wild-type rab5 independent of GTPgammaS.

Original publication

DOI

10.1091/mbc.9.2.323

Type

Journal article

Journal

Mol Biol Cell

Publication Date

02/1998

Volume

9

Pages

323 - 332

Keywords

Alkyl and Aryl Transferases, Androstadienes, Animals, Carrier Proteins, Cell Line, Clathrin, Coated Vesicles, Cricetinae, Endosomes, Enzyme Activation, Enzyme Inhibitors, GTP Phosphohydrolases, GTP-Binding Proteins, Guanosine 5'-O-(3-Thiotriphosphate), Kidney, Membrane Fusion, Mutation, Phosphatidylinositol 3-Kinases, Phosphoinositide-3 Kinase Inhibitors, Ribonucleotides, Wortmannin, rab GTP-Binding Proteins, rab5 GTP-Binding Proteins