NDLI: Inhibitors of V-ATPase Proton Transport Reveal Uncoupling Functions of Tether Linking Cytosolic and Membrane Domains of V0 Subunit a (Vph1p)

Content Provider	Journal of Biological Chemistry (JBC)
Author	Chan, Chun-Yuan Prudom, Catherine Raines, Summer M. Charkhzarrin, Sahba Melman, Sandra D. Haro, Leyma P. De Allen, Chris Lee, Samuel A. Sklar, Larry A. Parra, Karlett J.
Abstract	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 μm) and thonzonium bromide (EC50 = 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 V0 were resistant to thonzonium bromide but not to alexidine dihydrochloride, suggesting that this conserved sequence confers uncoupling potential to V1V0 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.
Related Links	http://www.jbc.org/content/287/13/10236.abstract
Ending Page	10250
Starting Page	10236
Page Count	15
File Format	HTM / HTML PDF
ISSN	00219258
Journal	Journal of Biological Chemistry (JBC)
Issue Number	13
Volume Number	287
DOI	10.1074/jbc.M111.321133
e-ISSN	1083351X
Language	English
Publisher	American Society for Biochemistry and Molecular Biology
Publisher Date	2012-03-23
Access Restriction	Open
Subject Keyword	ATPases H+-ATPase Membrane Proteins Proton Transport Vacuolar ATPase Yeast V-ATPase Coupling Inhibitors Uncouplers Membrane Biology
Content Type	Text
Resource Type	Article
Subject	Cell Biology Biochemistry Molecular Biology

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Inhibitors of V-ATPase Proton Transport Reveal Uncoupling Functions of Tether Linking Cytosolic and Membrane Domains of V0 Subunit a (Vph1p)*

The Tether Connecting Cytosolic (N Terminus) and Membrane (C Terminus) Domains of Yeast V-ATPase Subunit a (Vph1) Is Required for Assembly of V0 Subunit d

The tether connecting cytosolic (N terminus) and membrane (C terminus) domains of yeast V-ATPase subunit a (Vph1) is required for assembly of V0 subunit d.

The Tether Connecting Cytosolic (N Terminus) and Membrane (C Terminus) Domains of Yeast V-ATPase Subunit a (Vph1) Is Required for Assembly of V0 Subunit d*

The Amino-terminal Domain of the E Subunit of Vacuolar H+-ATPase (V-ATPase) Interacts with the H Subunit and Is Required for V-ATPase Function

The H Subunit (Vma13p) of the Yeast V-ATPase Inhibits the ATPase Activity of Cytosolic V1 Complexes

Cysteine-directed Cross-linking to Subunit B Suggests That Subunit E Forms Part of the Peripheral Stalk of the Vacuolar H+-ATPase

Evidence That the NH2 Terminus of Vph1p, an Integral Subunit of the V0 Sector of the Yeast V-ATPase, Interacts Directly with the Vma1p and Vma13p Subunits of the V1Sector

Structural Analysis of the N-terminal Domain of Subunit a of the Yeast Vacuolar ATPase (V-ATPase) Using Accessibility of Single Cysteine Substitutions to Chemical Modification

Inhibitors of V-ATPase Proton Transport Reveal Uncoupling Functions of Tether Linking Cytosolic and Membrane Domains of V0 Subunit a (Vph1p)

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