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© The Rockefeller University Press, 0021-9525/1997//283 $5.00
The Journal of Cell Biology, Volume 138, Number 2, , 1997 283-290

Article

A Novel Rab9 Effector Required for Endosome-to-TGN Transport



Elva Díaz, Frauke Schimmöller, and Suzanne R. Pfeffer

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.

This research was funded by a research grant to S.R. Pfeffer from the National Institutes of Health (DK37332), a predoctoral fellowship from the National Science Foundation to E. Díaz, and by a postdoctoral fellowship from the Human Frontier Science Program Organization and CIBA-Geigy Jubiläumsstiftung to F. Schimmöller.

Please address all correspondence to Suzanne R. Pfeffer, Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305-5307. Tel.: (650) 723-6169; Fax: (650) 725-6776; E-Mail: pfeffer{at}cmgm.stanford.edu

1. Abbreviation used in this paper: MPR, mannose 6-phosphate receptor.


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