RAB7 REGULATES TRANSPORT FROM EARLY TO LATE ENDOCYTIC COMPARTMENTS INXENOPUS OOCYTES

Rab7 has been shown to localize to late endosomes and to mediate trans port from early to late endosome/lysosome in mammalian cells and in ye ast. We developed a novel assay to quantify transport from early to la te endosomes using the Xenopus oocyte. Oocytes were pulsed with avidin after which the oocytes were incubated to allow avidin transport to a late compartment. The oocytes were then allowed to internalize biotin -horseradish peroxidase (HRP), The oocytes were then injected with tes t proteins and incubated further to allow transport of biotin-HRP from early endosomes to late endosomal/lysosomal compartments. Transport w as quantified by assessing the formation of HRP-biotin-avidin complexe s, Injection of Rab7:wild-type (WT) and Rab7:Q67L, a GTPase defective mutant, stimulated transport. RabB:WT had no effect. Rab7:WT-stimulate d transport was inhibited by nocodazole, suggesting a role for intact microtubules. Wortmannin, a phosphatidylinositol 3-kinase inhibitor, b locked Rab7:WT-stimulated transport, but Rab7:Q67L-stimulated transpor t was unaffected by the drug. Rab7:Q67L is constitutively activated an d may not require phosphatidylinositol 3-kinase activity for activatio n, Rab7-stimulated transport requires N-ethylmaleimide-sensitive facto r (NSF) activity as transport was blocked by N-ethyhmaleimide and ATPa se defective NSF mutants. Our results indicate that sequentially actin g endocytic Rab GTPases utilize similar factors although their modes o f action may be different.