Inhibitors of V-ATPase proton transport reveal uncoupling functions of tether linking cytosolic and membrane domains of V0 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 (EC(50) = 39 μM) and thonzonium bromide (EC(50) = 69 μM), 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 concentrations greater than 10 μM 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 Vph1p subunit a of V(0) were resistant to thonzonium bromide but not to alexidine dihydrochloride, suggesting that this conserved sequence confers uncoupling potential to V(1)V(0) complexes and that alexidine dihydrochloride uncouples the enzyme by a different mechanism. The inhibitors also uncoupled the Candida albicans enzyme and prevented cell growth, showing further specificity for V-ATPases. Thus, a new class of V-ATPase inhibitors (uncouplers), which are not simply ionophores, provided new insights into the enzyme mechanism and original evidence supporting the hypothesis that V-ATPases may not be optimally coupled in vivo. The consequences of uncoupling V-ATPases in vivo as potential drug targets are discussed.
Department of Biochemistry and Molecular Biology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131.
Chun-Yuan
CY
Chan
Center for Molecular Discovery, School of Medicine, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131.
Catherine
C
Prudom
Department of Biochemistry and Molecular Biology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131.
Summer M
SM
Raines
Department of Biochemistry and Molecular Biology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131.
Sahba
S
Charkhzarrin
Department of Biochemistry and Molecular Biology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131.
Sandra D
SD
Melman
Department of Biochemistry and Molecular Biology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131.
Leyma P
LP
De Haro
Center for Molecular Discovery, School of Medicine, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131.
Chris
C
Allen
Department of Internal Medicine, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131.
Samuel A
SA
Lee
Center for Molecular Discovery, School of Medicine, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131; Department of Pathology and Cancer Center, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131.
Larry A
LA
Sklar
Department of Biochemistry and Molecular Biology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131. Electronic address: kjparra@salud.unm.edu.
Karlett J
KJ
Parra
D001645:Biguanides / Q000494:pharmacology*; D002176:Candida albicans / Q000201:enzymology / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000201:enzymology; D009154:Mutation; D017434:Protein Structure, Tertiary; D018892:Proton-Motive Force / Q000187:drug effects* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D025262:Vacuolar Proton-Translocating ATPases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D014617:Vacuoles / Q000201:enzymology / Q000235:genetics
D001645:Biguanides; D004791:Enzyme Inhibitors; D029701:Saccharomyces cerevisiae Proteins; D025262:Vacuolar Proton-Translocating ATPases; C001570:alexidine