Rab11-like GTPase associates with and regulates the structure and functionof the contractile vacuole system in Dictyostelium

Screening of a cDNA library revealed the existence of a Dictyostelium cDNA encoding a protein 80% identical at the amino acid level to mammalian Rab11 . Subcellular fractionation and immunofluorescence studies revealed that Dd Rab11 was exclusively associated with the ATPase proton pump-rich contracti le vacuole membrane system, consisting of a reticular network and bladder-l ike vacuoles. Video microscopy of cells expressing GFP-DdRab11 revealed tha t this Rab was associated with contractile vacuolar bladders undergoing for mation, fusion and expulsion of water. The association of DdRab11 with cont ractile vacuole membranes was disrupted when cells were exposed to either h ypo-osmotic conditions or an inhibitor of the ATPase proton pump. Cells tha t overexpressed a dominant negative form of DdRab11 were analyzed biochemic ally and microscopically to measure changes in the structure and function o f the contractile vacuole system. Compared with wild-type cells, the domina nt negative DdRab11-expressing cells contained a more extensive contractile vacuole network and abnormally enlarged contractile vacuole bladders, most likely the result of defects in membrane trafficking. In addition, the mut ant cells enlarged, detached from surfaces and contained large vacuoles whe n exposed to water, suggesting a functional defect in osmotic regulation. N o changes were observed in mutant cells in the rate of fluid phase internal ization or release, suggesting the DdRab11-mediated membrane trafficking de fects were not general in nature. Surprisingly, the rate of phagocytosis wa s increased in the dominant negative DdRab11-expressing cells when compared with control cells. Our results are consistent with a role for DdRab11 in regulating membrane traffic to maintain the normal morphology and function of the contractile vacuole.