We have identified and characterized two proton-pumping ATPases in the fungus Neurospora crassa, one in the plasma membrane, another in the vacuolar membrane. The long term goal of our research is to describe the mechanism by which these enzymes translocate protons and to understand their genetics and kinetic regulation. The plasma membrane ATPase is very similar to the (Na+,K+)-ATPase of animal cell membranes and the Ca++ ATPase of sarcoplasmic reticulum. The purified enzyme has a single type of polypeptide (104,000 MW) but the number of polypetides per functional unit is unknown. The kinetic behavior of the ATPase strongly suggests a dimeric structure with interacting subunits. We propose to test this hypothesis by direct binding studies to measure the number and the affinity of the active sites, by size determination using radiation inactivation, by attempting to monormerize the enzyme with the detergent C12E8, and by probing the enzyme with fluorescent affinity lables for the substrate-binding sites. A second major project with the plasma membrane ATPase is the isolation of mutant forms of the enzyme. We have devised a selection scheme which yields strains with low levels of ATPase activity, some of which altered in the 104,000 MW polypeptide of the ATPase. characterization of these mutants is an important new approache to understanding the mechanism and regulation of the enzyme. The vauolar membrane ATPase, in its substrate affinity and substrate specificity, resembles the mitochondrial ATPase, suggesting it may be an FOF1-type enzyme. Studied with mutant strains altered in the mitochondrial ATPase have firmly established. however, that the vacuolar ATPase and thus describe its polypeptide composition. We are also using vacuolar membrane vesicles to demonstrate that the enzyme functions as a proton pump, driving the active transport of basic amino acids into the vacuole. If the vacuolar ATPase is similar to the mitochondrial ATPase we might expect immunological crossreactivity. Using antibodies to F1 ATPases from Neurospora and E. coli we find that specific vacuolar polypeptides do crossreact. Preliminary experiments, which we plan to pursue, indicate that the plama membrane, vacuolar membrane, and mitochondrial membrane ATPases may all be related.
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