9728403 Clarke Abstract All eukaryotic cells contain a multi-subunit enzyme called the vacuolar proton pump or V-ATPase which transports protons across cellular membranes. In mammals this enzyme is needed for kidney function, uptake of neurotransmitter molecules, acidification of intracellular compartments such as endosomes and lysosomes, and bone resorption. The present study uses a simple amoeboid cell, Dictyostelium, to explore the functions of the V-ATPase. These cells contain a V-ATPase similar to that in mammals. The pump is enriched in a specialized organelle called the contractile vacuole complex, which sequesters and then expels excess fluid from the cell, much as the kidney does in higher organisms. Using Dictyostelium cells, it is possible to explore the functions of the V-ATPase by altering the genes that encode its subunits. The gene for a large transmembrane subunit of the V-ATPase, whose function is poorly understood, has been cloned. Attempts to disrupt this gene (vatM) or to block its function by antisense mutagenesis have not been successful and argue that this is an essential gene. However, a conditional mutant has been created by replacing the promotor of the gene with a different promotor to change how the expression of the gene is regulated. Under conditions that greatly reduce the expression of this mutant gene, cells that carry the gene grow poorly and mislocalize other proton pump subunits. These mutant cells, deficient in active proton pumps, will be used to determine the roles of the V-ATPase in pH homeostasis and the acidification of the endosomal and lysosomal compartments in Dictyostelium. Antibody probes for proton pump subunits and for other components of the contractile vacuoles and endosomes will be used to monitor the organization of these compartments and their interrelationships. In addition, fluorescent versions of these proteins will be prepared as tracers to observe the dynamics of these compartments in living cells. In other experiment s, the redistribution of proton pumps from internal compartments to the plasma membrane will be examined since this also occurs in mammalian cells where these pumps are found. The studies as a whole will explore several important features and functions of the V-ATPase in a model cell system that resembles mammalian cells but is more amenable to molecular genetic manipulation.
