Sequential actions of Rab5 and Rab7 regulate endocytosis in the Xenopus oocyte.
To explore the role of GTPases in endocytosis, we developed an assay using Xenopus oocytes injected with recombinant proteins to follow the uptake of the fluid phase marker HRP. HRP uptake was inhibited in cells injected with GTPgammaS or incubated with aluminum fluoride, suggesting a general role for GTPases in endocytosis. Injection of Rab5 into oocytes, as well as Rab5:Q79L, a mutant with decreased GTPase activity, increased HRP uptake. Injection of Rab5:S34N, the dominant-negative mutant, inhibited HRP uptake. Injection of N-ethylmaleimide-sensitive factor (NSF) stimulated HRP uptake, and ATPase-defective NSF mutants inhibited HRP uptake when coinjected with Rab5:Q79L, confirming a requirement for NSF in endocytosis. Surprisingly, injection of Rab7:WT stimulated both uptake and degradation/activation of HRP. The latter appears to be due to enhanced transport to a late endosomal/prelysosomal degradative compartment that is monensin sensitive. Enhancement of uptake by Rab7 appears to function via an Rab5-sensitive pathway in oocytes since the stimulatory effect of Rab7 was blocked by coinjection of Rab5:S34N. Stimulation of uptake by Rab5 was blocked by Rab5:S34N but not by Rab7:T22N. Our results suggest that Rab7, while functioning downstream of Rab5, may be rate limiting for endocytosis in oocytes.
Mukhopadhyay
A
A
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Barbieri
A M
AM
Funato
K
K
Roberts
R
R
Stahl
P D
PD
D000818:Animals; D002352:Carrier Proteins / Q000502:physiology*; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D005260:Female; D019204:GTP-Binding Proteins / Q000235:genetics / Q000502:physiology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000378:metabolism; D008845:Microinjections; D008985:Monensin / Q000494:pharmacology; D052066:N-Ethylmaleimide-Sensitive Proteins; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D017354:Point Mutation; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D033921:Vesicular Transport Proteins; D014982:Xenopus laevis; D020691:rab GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins; D000090802:rab7 GTP-Binding Proteins
D002352:Carrier Proteins; D011993:Recombinant Fusion Proteins; D033921:Vesicular Transport Proteins; D000090802:rab7 GTP-Binding Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D008985:Monensin; D006735:Horseradish Peroxidase; D019204:GTP-Binding Proteins; D052066:N-Ethylmaleimide-Sensitive Proteins; D020691:rab GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins