Abstract
Rab22a is a member of the Rab family of small GTPases that localizes in the endocytic pathway. In CHO cells, expression of canine Rab22a (cRab22a) causes a dramatic enlargement of early endocytic compartments. We wondered whether transferrin recycling is altered in these cells. Expression of the wild-type protein and a GTP hydrolysis-deficient mutant led to the redistribution of transferrin receptor to large cRab22a-positive structures in the periphery of the cell and to a significant decrease in the plasma membrane receptor. Kinetic analysis of transferrin uptake indicates that internalization and early recycling were not affected by cRab22a expression. However, recycling from large cRab22a-positive compartments was strongly inhibited. A similar effect on transferrin transport was observed when human but not canine Rab22a was expressed in HeLa cells. After internalization for short periods of time (5 to 8 min) or at a reduced temperature (16°C), transferrin localized with endogenous Rab22a in small vesicles that did not tubulate with brefeldin A, suggesting that the endogenous protein is present in early/sorting endosomes. Rab22a depletion by small interfering RNA disorganized the perinuclear recycling center and strongly inhibited transferrin recycling. We speculate that Rab22a controls the transport of the transferrin receptor from sorting to recycling endosomes.
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This work was partly supported by an International Research Scholar Award from the Howard Hughes Medical Institute and by a grant from ANPCyT, Argentina, to L.S.M. and by NIH GM 042259 and the NSF U.S.-Argentina Cooperative Research Award to P.D.S. J.G.M. is a CONICET fellow (Argentina) and has received support from IUBMB and the Journal of Cell Science for training visits to the P.D.S. laboratory.
We thank E. Peters, A. Medero, and M. Furlán for excellent technical assistance, J. Gruenberg and W. Brown for antibodies, and M. Zerial, I. Mellman, M. I. Colombo, and T. Galli for plasmids. We thank M. I. Colombo and M. T. Damiani for critical reading of and helpful comments on the manuscript.