Inhibitors of V-ATPase Proton Transport Reveal Uncoupling Functions of the Tether Linking Cytosolic and Membrane Domains of the Vo subunit a (Vph1p)

Vacuolar ATPases (V-ATPases) are important for many cellular processes, as they regulate pH by pumping cytosolic protons into intracellular organelles. The cytoplasm is acidified when V-ATPase is inhibited; thus we conducted a high-throughput screen of a chemical library to search for compounds that acidify the yeast cytosol in vivo using pHLuorin-based flow cytometry. Two inhibitors, alexidine dihydrochloride (EC50=39 M) and thonzonium bromide (EC50=69 M), prevented ATP-dependent proton transport in purified vacuolar membranes. They acidified the yeast cytosol and caused pH-sensitive growth defects typical of V-ATPase mutants (vma phenotype). At greater than 10 M concentrations the inhibitors were cytotoxic, even at the permissive pH (pH 5.0). Membrane fractions treated with alexidine dihydrochloride and thonzonium bromide fully retained concanamycin A-sensitive ATPase activity despite the fact that proton translocation was inhibited by 80-90%, indicating that V-ATPases were uncoupled. Mutant V-ATPase membranes lacking residues 362-407 of the tether of the Vph1p subunit a of Vo were resistant to thonzonium bromide but not to alexidine dihydrochloride, suggesting that this conserved sequence confers uncoupling potential to V1Vo complexes and that alexidine dihydrochloride uncouple the enzyme by a different mechanism. The inhibitors also uncoupled the Candida albicans enzyme and prevented cell growth, further showing specificity for V-ATPases. Thus, a new class of V-ATPase inhibitor (uncouplers) which are not simply ionophores, provided new insights into the enzyme mechanism and http://www.jbc.org/cgi/doi/10.1074/jbc.M111.321133 The latest version is at JBC Papers in Press. Published on January 3, 2012 as Manuscript M111.321133 Copyright 2012 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on A uust 9, 2017 hp://w w w .jb.org/ D ow nladed from