| The contractile vacuole (CV) system is the osmoregulatory organelle of free-living amoebae and protozoa. I present data showing that the RabGAP RabGAP1 acts as a switch for discharging the CVs into the extracellular medium in Dictyostelium. rabgap1 null (rabgap1-) cells have highly enlarged CVs whose structure and activity are aberrant. In rabgap1- cells, the dynamic fusion of the CV with the plasma membrane is absent and the discharge of CV content is inefficient. RabGAP1 localizes to the CV membrane whereupon the vacuoles stop charging, become round, and fuse to the plasma membrane. Drainin, another TBC-domain containing protein, and RabGAP1 sequentially localize to the CV membrane and regulate CV discharge. Rab8A and Rab11A interact with RabGAP1, co-localize with RabGAP1 on the CV, and suppress the rabgap1- large vacuole phenotype. However, Rab8A suppresses both the large vacuole phenotype and the abnormal discharging phenotype in drainin - cells. I identified two BEACH family proteins, LvsA and LvsD, as a suppressor and an enhancer of the rabgap1- large CV phenotype, respectively. Analysis of LvsA and LvsD mutant strains in a rabgap1- background revealed that LvsA and LvsD have distinct functions in regulating CV biogenesis by controlling the recycling of CV membranes. My studies help define the pathways controlling CV regulation and biogenesis.; In the last part of the dissertation, I provide evidence that CVs might be the intracellular storage of Ca2+ to regulate F-actin polymerization and chemotaxis in Dictyostelium. |
