Regulators of membrane fusion in Dictyostelium discoideum

The Rab GTPases are small molecular weight (21–25 kDa) molecular switches that regulate intracellular membrane trafficking in eukaryotic cells. The genetically amenable single-celled eukaryote, Dictyostelium discoideum , is an excellent model organism to study membrane trafficking because it displays high rates of phagocytosis, endocytosis, and endosomal membrane flow. The Rab proteins are thought to be involved with many of the steps of membrane trafficking including vesicle budding, targeting, docking, and fusion. Homotypic fusion of lysosomes results in the intermixing of lumenal contents and heterotypic fusion in which lysosomes filled with hydrolases fuse with phagosomes is necessary to deliver enzymes needed to kill the internalized pathogen. Our current studies indicate that RabD, a Rab14-like GTPase, is involved with the fusion of macropinosomes, lysosomes, and phagosomes. Cells expressing dominant negative RabD were deficient in macropinocytosis, delayed in lysosome fusion to form post-lysosomes, and deficient in phagosome-phagosome fusion and internalization of particles. RabD may regulate membrane fusion via a downstream mediator, PI 3-kinase. LvsB, the orthologue of the Lyst protein that is involved with Chediak-Higashi Syndrome, is a negative regulator of lysosome-lysosome fusion and may counterbalance the activities of RabD. It is not known if RabD and LvsB directly interact, though it is more likely that a common downstream effector may balance the fusion/fission activities of these two molecules. Other GTPases that partially colocalize with RabD include RabB and Rab11a. The dominant negative expression of RabB, a Rab21-like GTPase, significantly decreased the rate of phagocytosis with a concomitant slight decrease in fluid phase endocytosis. Rab11a is predominantly localized to the contractile vacuole (CV), an osmoregulatory organelle, and is involved with the homotypic fusion/fission reactions and maintenance of those membranes. Surprisingly, Rab11a also acts as a negative regulator of phagocytosis, possibly having to do with supplying membrane to the forming phagocytic cup.