A ROLE FOR A RAB4-LIKE GTPASE IN ENDOCYTOSIS AND IN REGULATION OF CONTRACTILE VACUOLE STRUCTURE AND FUNCTION IN DICTYOSTELIUM-DISCOIDEUM

The small M(r) Rab4-like GTPase, RabD, localizes to the endosomal path way and the contractile vacuole membrane system in Dictyostelium disco ideum. Stably transformed cell lines overexpressing a dominant negativ e functioning RabD internalized fluid phase marker at 50% of the rate of wild-type cells. Mutant cells were also slower at recycling interna lized fluid. Microscopic and biochemical approaches indicated that the transport of fluid to large postlysosome vacuoles was delayed in muta nt cells, resulting in an accumulation in acidic smaller vesicles, pro bably lysosomes. Also, RabD N121I-expressing cell lines missorted a sm all but significant percentage of newly synthesized lysosomal alpha-ma nnosidase precursor polypeptides. However, the majority of the newly s ynthesized alpha-mannosidase was transported with normal kinetics and correctly delivered to lysosomes. Subcellular fractionation and immuno fluorescent microscopy indicated that in mutant cells contractile vacu ole membrane proteins were associated with compartments morphologicall y distinct from the normal reticular network. Osmotic tests revealed t hat the contractile vacuole functioned inefficiently in mutant cells. Our results suggest that RabD regulates membrane traffic along the end osomal pathway, and that this GTPase may play a role in regulating the structure and function of the contractile vacuole system by facilitat ing communication with the endosomal pathway.