GDP dissociation inhibitor domain II required for Rab GTPase recycling

Rab GTPases are localized to distinct subsets of organelles within the cell , where they regulate SNARE-mediated membrane trafficking between organelle s. One factor required for Rab localization and function is Rab GDP dissoci ation inhibitor (GDI), which is proposed to recycle Rab after vesicle fusio n by extracting Rab from the membrane and loading Rab onto newly formed tra nsport intermediates. GDI is composed of two domains; Rab binding is mediat ed by Domain I, and the function of Domain II is not known, In this study, Domain II of yeast GDI, encoded by the essential GDI1/SEC19 gene, was targe ted in a genetic screen to obtain mutants that might lend insight into the function of this domain. In one gdi1 mutant, the cytosolic pools of all Rab s tested were depleted, and Rab accumulated on membranes, suggesting that t his mutant Gdi1 protein has a general defect in extraction of Rab from memb ranes, In a second gdi1 mutant, the endosomal/vacuolar Rabs Vps21/Ypt51p an d Ypt7p accumulated in the cytosol bound to Gdi1p, but localization of Ypt1 p and Sec4p were not significantly affected. Using an in vitro assay which reconstitutes Gdi1p-mediated membrane loading of Rab, this mutant Gdi1p was found to be defective in loading of Vps21p but not Ypt1p, Loading of Vps21 p by loading-defective Gdi1p was restored when acceptor membranes prepared from a deletion strain lacking Vps21p were used. These results suggest that membrane-associated Rab may regulate recruitment of GDI-Rab from the cytos ol, possibly by regulating a GDI-Rab receptor. We conclude that Domain II o f Gdi1p is essential for Rab loading and Rab extraction, and confirm that e ach of these activities is required for Gdi1p function in vivo.