Rab22a affects the morphology and function of the endocytic pathway

Soon after endocytosis, internalized material is sorted along different pat hways in a process that requires the coordinated activity of several Rab pr oteins. Although abundant information is available about the subcellular di stribution and function of some of the endocytosis-specific Rabs (e.g. Rab5 and Rab4), very little is known about some other members of this family of proteins. To unveil some of the properties of Rab22a, one of the less stud ied endosome-associated small GTPases, we have expressed the protein tagged with the green fluorescent protein in CHO cells. The results indicate that Rab22a associates with early and late endosomes (labeled by a 5 minute rho damine-transferrin uptake and the cation-independent mannose 6-phosphate re ceptor, respectively) but not with lysosomes (labeled by 1 hour rhodamine h orseradish peroxidase uptake followed by 1 hour chase). Overexpression of t he protein causes a prominent morphological enlargement of the early and la te endosomes. Two mutants were generated by site-directed mutagenesis, a ne gative mutant (Rab22aS19N, with reduced affinity for GTP) and a constitutiv ely active mutant (Rab22aQ64L, with reduced endogenous GTPase activity). Th e distribution of the negative mutant was mostly cytosolic, whereas the pos itive mutant associated with early and late endosomes and, interestingly al so with lysosomes and autophagosomes (labeled with monodansylcadaverine). C ells expressing Rab22a wild type and Rab22aS19N displayed decreased endocyt osis of a fluid phase marker. Conversely, overexpression of Rab22aQ64L, whi ch strongly affects the morphology of endosomes, did not inhibit bulk endoc ytosis. Our results show that Rab22a has a unique distribution along the en docytic pathway that is not shared by any other Rab protein, and that it st rongly affects the morphology and function of endosomes.