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Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305-5307
Rab9 GTPase is required for the transport of
mannose 6-phosphate receptors from endosomes to
the trans-Golgi network in living cells, and in an in vitro
system that reconstitutes this process. We have used the
yeast two-hybrid system to identify proteins that interact preferentially with the active form of Rab9. We report here the discovery of a 40-kD protein (p40) that
binds Rab9-GTP with roughly fourfold preference to
Rab9-GDP. p40 does not interact with Rab7 or K-Ras;
it also fails to bind Rab9 when it is bound to GDI.
The protein is found in cytosol, yet a significant fraction (~30%) is associated with cellular membranes.
Upon sucrose density gradient flotation, membrane-
associated p40 cofractionates with endosomes containing mannose 6-phosphate receptors and the Rab9 GTPase. p40 is a very potent transport factor in that the pure, recombinant protein can stimulate, significantly,
an in vitro transport assay that measures transport of
mannose 6-phosphate receptors from endosomes to the
trans-Golgi network. The functional importance of p40
is confirmed by the finding that anti-p40 antibodies inhibit in vitro transport. Finally, p40 shows synergy with
Rab9 in terms of its ability to stimulate mannose 6-phosphate receptor transport. These data are consistent
with a model in which p40 and Rab9 act together to
drive the process of transport vesicle docking.
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