Rab7 regulates transport from early to late endocytic compartments in Xenopus oocytes.
Rab7 has been shown to localize to late endosomes and to mediate transport from early to late endosome/lysosome in mammalian cells and in yeast. We developed a novel assay to quantify transport from early to late 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 test proteins and incubated further to allow transport of biotin-HRP from early endosomes to late endosomal/lysosomal compartments. Transport was quantified by assessing the formation of HRP-biotin-avidin complexes. Injection of Rab7:wild-type (WT) and Rab7:Q67L, a GTPase defective mutant, stimulated transport. Rab5:WT had no effect. Rab7:WT-stimulated transport was inhibited by nocodazole, suggesting a role for intact microtubules. Wortmannin, a phosphatidylinositol 3-kinase inhibitor, blocked Rab7:WT-stimulated transport, but Rab7:Q67L-stimulated transport was unaffected by the drug. Rab7:Q67L is constitutively activated and may not require phosphatidylinositol 3-kinase activity for activation. Rab7-stimulated transport requires N-ethylmaleimide-sensitive factor (NSF) activity as transport was blocked by N-ethylmaleimide and ATPase defective NSF mutants. Our results indicate that sequentially acting endocytic Rab GTPases utilize similar factors although their modes of action may be different.
Mukhopadhyay
A
A
Washington University School of Medicine, Department of Cell Biology & Physiology, St. Louis, Missouri 63110, USA.
Funato
K
K
Stahl
P D
PD
D000818:Animals; D001681:Biological Assay / Q000379:methods; D001692:Biological Transport; D011992:Endosomes / Q000378:metabolism*; D005260:Female; D019204:GTP-Binding Proteins / Q000378:metabolism*; D009865:Oocytes / Q000378:metabolism* / Q000648:ultrastructure; D014981:Xenopus; D020691:rab GTP-Binding Proteins; D000090802:rab7 GTP-Binding Proteins
D000090802:rab7 GTP-Binding Proteins; D019204:GTP-Binding Proteins; D020691:rab GTP-Binding Proteins