Isolation and initial characterization of the bipartite contractile vacuole complex from Dictyostelium discoideum

• Published in: The Journal of biological chemistry 1994-01, Vol.269 (3), p.2225-2233
• Main Authors: NOLTA, K. V, STECK, T. L
• Format: Article
• Language: English
• Subjects: Acid Phosphatase - analysis; Alkaline Phosphatase - analysis; Animals; Biological and medical sciences; Biomarkers - analysis; Body Water - metabolism; Cell Fractionation - methods; Cell physiology; Centrifugation, Density Gradient; Dictyostelium - physiology; Dictyostelium - ultrastructure; Dictyostelium discoideum; Fundamental and applied biological sciences. Psychology; Histocytochemistry; Microscopy, Electron; Molecular and cellular biology; Phosphoric Diester Hydrolases - analysis; Proton-Translocating ATPases - metabolism; Secretion. Exocytosis; Vacuoles - physiology; Vacuoles - ultrastructure
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(17)42157-0

The contractile vacuole complex serves to excrete excess cytosolic water from protists. In the amoeba, Dictyostelium discoideum, the organelle had a bipartite morphology: a large main vacuole (bladder) marked by lumenal alkaline phosphatase was surrounded by numerous satellite vacuoles (spongiomes). Bladders and spongiomes have now been purified for the first time. The spongiome membranes had a high density of surface projections identified as catalytically-active vacuolar proton pumps (V-H(+)-ATPase). Spongiomes were resolved from the pump-poor bladders by 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 bladders and populated the proton pump-rich membrane fraction called acidosomes. Isolated bladders were enriched > 40-fold in alkaline phosphatase and phosphodiesterase, the activities of which were > 85% latent. Bladders depleted of spongiomes bore several distinctive polypeptides; they also had an excess of the basepieces of the proton pump over the catalytic heads. Bladder membranes were also lipid-rich and had a distinctive lipid composition. We conclude that the contractile vacuole system in Dictyostelium is a complex of discrete, separable bladder and spongiome membranes. The V-H(+)-ATPase in the spongiome may catalyze the primary energy transduction step for pumping water out of the cytoplasm.