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Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110
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 GTP
S 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.
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