MUTATIONS OF PMA-1, THE GENE ENCODING THE PLASMA-MEMBRANE H-ATPASE OFNEUROSPORA-CRASSA, SUPPRESS INHIBITION OF GROWTH BY CONCANAMYCIN-A, ASPECIFIC INHIBITOR OF VACUOLAR ATPASES()

Concanamycin A (CCA), a specific inhibitor of vacuolar ATPases, inhibi ted growth of Neurospora crassa in medium adjusted to pH 7 or above. M utant strains were elected for growth on medium containing 1.0 mu M CC A. Sixty-four (of 66) mutations mapped in the region of the pma1 locus , which encodes the plasma membrane H+- ATPase. Analysis of V-ATPase a ctivity in isolated vacuolar membranes from the mutant strains showed wildtype activity and sensitivity to CCA. In contrast, plasma membrane H+-ATPase activity in isolated plasma membranes from the mutants was reduced as compared with wild-type, and in four strains the activity s howed increased resistance to vanadate. The most interesting change in the plasma membrane H+-ATPase was in kinetic behavior. The wild-type enzyme showed sigmoid dependence on MgATP concentration with a Hill nu mber of 2.0, while the seven mutants tested exhibited hyperbolic kinet ics with a Hill number of 1.0. One interpretation of these data was th at the enzyme had changed from a functional dimer to a functional mono mer. Mutation of the plasma membrane H+-ATPase did not confer resistan ce by preventing uptake of CCA. In the presence of CCA both wild-type and mutant strains were unable to accumulate arginine, failed to conce ntrate chloroquine in acidic vesicles, and exhibited gross alterations in hyphal morphology, indicating that the CCA had entered the cells a nd inactivated the V-ATPase. Instead, we hypothesize that the mutation s conferred resistance because the altered plasma membrane H+- ATPase could more efficiently rid the cell of toxic levels of Ca2+ or protons or other ions accumulated in the cytoplasm following inactivation of the V-ATPase by CCA.