ArticleAnalytical biochemistry2010
Identification of inhibitors of vacuolar proton-translocating ATPase pumps in yeast by high-throughput screening flow cytometry.
Article in Analytical biochemistry, 2010. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 22 papers.
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Who cites it
22 citing papers in PubMed, 34 citations in OpenAlex.
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- Neurodegeneration-related beta-amyloid as autocatabolism-attenuator in aIBRO reports · 2020Article
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- Vacuolar proton-translocating ATPase is required for antifungal resistance and virulence of Candida glabrata.PloS one · 2019Article
- Identification of small molecules that disrupt vacuolar function in the pathogen Candida albicans.PloS one · 2017Article
- Affinity Purification and Structural Features of the Yeast Vacuolar ATPase Vo Membrane Sector.The Journal of biological chemistry · 2015Article
- Regulation of lipid droplet dynamics in Saccharomyces cerevisiae depends on the Rab7-like Ypt7p, HOPS complex and V1-ATPase.Biology open · 2015Article
- Experimental and computational analysis of a large protein network that controls fat storage reveals the design principles of a signaling network.PLoS computational biology · 2015Article
- Disulfiram attenuates osteoclast differentiation in vitro: a potential antiresorptive agent.PloS one · 2015Article
- Yeast phosphofructokinase-1 subunit Pfk2p is necessary for pH homeostasis and glucose-dependent vacuolar ATPase reassembly.The Journal of biological chemistry · 2014Article
- A high throughput flow cytometric assay platform targeting transporter inhibition.Drug discovery today. Technologies · 2014Review
- A functional screen for copper homeostasis genes identifies a pharmacologically tractable cellular system.BMC genomics · 2014Article
- Advances in targeting the vacuolar proton-translocating ATPase (V-ATPase) for anti-fungal therapy.Frontiers in pharmacology · 2014Review
- Essential role for vacuolar acidification in Candida albicans virulence.The Journal of biological chemistry · 2013Article
- Measurement of vacuolar and cytosolic pH in vivo in yeast cell suspensions.Journal of visualized experiments : JoVE · 2013Article
- Estimating the rotation rate in the vacuolar proton-ATPase in native yeast vacuolar membranes.European biophysics journal : EBJ · 2013Article
- Inhibitors of V-ATPase proton transport reveal uncoupling functions of tether linking cytosolic and membrane domains of V0 subunit a (Vph1p).The Journal of biological chemistry · 2012Article
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Authors and funding
7 authors at 2 institutions in 1 country.
Funding
Abstract
Fluorescence intensity of the pH-sensitive carboxyfluorescein derivative 2,7-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) was monitored by high-throughput flow cytometry in living yeast cells. We measured fluorescence intensity of BCECF trapped in yeast vacuoles, acidic compartments equivalent to lysosomes where vacuolar proton-translocating ATPases (V-ATPases) are abundant. Because V-ATPases maintain a low pH in the vacuolar lumen, V-ATPase inhibition by concanamycin A alkalinized the vacuole and increased BCECF fluorescence. Likewise, V-ATPase-deficient mutant cells had greater fluorescence intensity than wild-type cells. Thus, we detected an increase of fluorescence intensity after short- and long-term inhibition of V-ATPase function. We used yeast cells loaded with BCECF to screen a small chemical library of structurally diverse compounds to identify V-ATPase inhibitors. One compound, disulfiram, enhanced BCECF fluorescence intensity (although to a degree beyond that anticipated for pH changes alone in the mutant cells). Once confirmed by dose-response assays (EC(50)=26 microM), we verified V-ATPase inhibition by disulfiram in secondary assays that measured ATP hydrolysis in vacuolar membranes. The inhibitory action of disulfiram against V-ATPase pumps revealed a novel effect previously unknown for this compound. Because V-ATPases are highly conserved, new inhibitors identified could be used as research and therapeutic tools in cancer, viral infections, and other diseases where V-ATPases are involved.
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