SEQUENTIAL ACTIONS OF RAB5 AND RAB7 REGULATE ENDOCYTOSIS IN THE XENOPUS OOCYTE

To explore the role of GTPases in endocytosis, we developed an assay u sing Xenopus oocytes injected with recombinant proteins to follow the uptake of the fluid phase marker HRP. HRP uptake was inhibited in cell s injected with GTP gamma S or incubated with aluminum fluoride, sugge sting a general role for GTPases in endocytosis. Injection of Rab5 int o oocytes, as well as Rab5:Q79L, a mutant with decreased GTPase activi ty, increased HRP uptake. Injection of Rab5:S34N, the dominant-negativ e mutant, inhibited HRP uptake. Injection of N-ethylmaleimide-sensitiv e factor (NSF) stimulated HRP uptake, and ATPase-defective NSF mutants inhibited HRP uptake when coinjected with Rab5:Q79L, confirming a req uirement for NSF in endocytosis, Surprisingly, injection of Rab7:WT st imulated both uptake and degradation/activation of HRP. The latter app ears to be due to enhanced transport to a late endosomal/prelysosomal degradative compartment that is monensin sensitive. Enhancement of upt ake by Rab7 appears to function via an Rab5-sensitive pathway in oocyt es 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 funct;nn;nn dow nstream of Rab5, may be rate limiting for endocytosis in oocytes.