ISOLATION AND INITIAL CHARACTERIZATION OF THE BIPARTITE CONTRACTILE VACUOLE COMPLEX FROM DICTYOSTELIUM-DISCOIDEUM

The contractile vacuole complex serves to excrete excess cytosolic wat er from protists. In the amoeba, Dictyostelium discoideum, the organel le had a bipartite morphology: a large main vacuole (bladder) marked b y lumenal alkaline phosphatase was surrounded by numerous satellite va cuoles (spongiomes). Bladders and spongiomes have now been purified fo r the first time. The spongiome membranes had a high density of surfac e projections identified as catalytically-active vacuolar proton pumps (V-H+-ATPase). Spongiomes were resolved from the pump-poor bladders b y immunogold buoyant density shift with antibodies to the V-H+-ATPase; they contained little protein other than this pump. It appears that, following homogenization, most of the spongiome dissociated from bladd ers and populated the proton pump-rich membrane fraction called acidos omes. Isolated bladders were enriched >40-fold in alkaline phosphatase and phosphodiesterase, the activities of which were >85% latent. Blad ders depleted of spongiomes bore several distinctive polypeptides; the y also had an excess of the basepieces of the proton pump over the cat alytic heads. Bladder membranes were also lipid-rich and had a distinc tive lipid composition. We conclude that the contractile vacuole syste m in Dictyostelium is a complex of discrete, separable bladder and spo ngiome membranes. The V-H+-ATPase in the spongiome may catalyze the pr imary energy transduction step for pumping water out of the cytoplasm.