INHIBITION OF ENDOSOME FUSION BY WORTMANNIN PERSISTS IN THE PRESENCE OF ACTIVATED RAB5

Rab5-dependent endosome fusion is sensitive to the phosphoinositide 3- kinase inhibitor, wortmannin. It has been proposed that phosphoinositi de 3-kinase activity may be required for activation of rab5 by influen cing its nucleotide cycle such as to promote its active GTP state. In this report we demonstrate that endosome fusion remains sensitive to w ortmannin despite preloading of endosomes with stimulatory levels of a GTPase-defective mutant rab5(Q79L) or of a xanthosine triphosphate-bi nding mutant, rab5(D136N), in the presence of the nonhydrolysable anal ogue XTP gamma S. These results suggest that activation of rab5 cannot be the principal function of the wortmannin-sensitive factor on the e ndosome fusion pathway. This result is extrapolated to all GTPases by demonstrating that endosome fusion remains wortmannin sensitive despit e prior incubation with the nonhydrolysable nucleotide analogue GTP ga mma S. Consistent with these results, direct measurement of clathrin-c oated vesicle-stimulated nucleotide dissociation from exogenous rab5 w as insensitive to the presence of wortmannin. A large excess of rab5(Q 79L), beyond levels required for maximal stimulation of the fusion ass ay, afforded protection against wortmannin inhibition, and partial pro tection was also observed with an excess of wild-type rab5 independent of GTP gamma S.