The activity of the vacuolar H(+)-ATPase has been characterized in isolated vacuoles of the yeast Saccharomyces cerevisiae by means of the patch-clamp technique. With cytosolic calcium at virtually zero (<10(-9) M), Mg-ATP induced a transient, bafilomycin A(1)-sensitive current corresponding to the flow of positive charges from the cytoplasmic surface to the vacuolar lumen. The Mg-ATP-dependent current reached its maximum amplitude (30+/-8 mA m(-2) with 5 mM Mg-ATP, n=34) within 15-20 s and declined slowly over a period of about 15-20 min even in the continuous presence of Mg-ATP. This decline of pumping activity was independent of the cytosolic KCl concentration, suggesting an inhibitory mechanism different from the high salt-induced dissociation of V(0) and V(1) reported for the V-ATPase of plants and fungi. Cytosolic ADP was found to modulate the pump activity since Mg-ATP-induced pump current was smaller if monitored in the presence of 5 mM ADP and addition of 5 mM ADP in the presence of 5 mM Mg-ATP reduced the pump current by more than 50%. Furthermore, reduction of the cytosolic ADP concentration by the ATP-regenerating system creatine phosphate/creatine kinase partially relieved the endogenous inhibition of the V-ATPase, confirming that interaction of cytosolic ADP with the V-ATPase is the reason for the transient nature of the pump current in yeast vacuoles.
|Evidence ID||Analyze ID||Interactor||Interactor Systematic Name||Interactor||Interactor Systematic Name||Type||Assay||Annotation||Action||Modification||Phenotype||Source||Reference||Note|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Reference||Annotation Extension|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Conditions||Strain||Source||Reference|