A high-throughput assay for mitochondrial membrane potential in permeabilized yeast cells.
A fluorometric assay for mitochondrial membrane potential in permeabilized yeast cells has been developed. This method involves permeabilizing the plasma membrane and measuring the distribution of a mitochondrial membrane potential sensitive probe 3,3'-dipropylthiadicarbocyanine iodide (DiSC(3)(5); DiSC(3)). In permeabilized cells, DiSC(3) fluorescence decreased when introduced into energized mitochondria and increased three- to sixfold when the mitochondrial membrane potential was dissipated by the chemical uncoupler carbonylcyanide m-chlorophenyl hydrazone. Plasma membrane potential was abolished by permeabilization, as shown by a lack of polarization of the plasma membrane induced by K(+) and glucose. Uncoupling protein 1 (UCP1), a mitochondrial H(+) transporter, was used as a model for method validation. The fluorescence intensity responded vigorously to specific modulators in UCP1-expressing cells. This method has been adapted as a high-throughput assay to screen for modulators of mitochondrial membrane potential.
Farrelly
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Tularik Inc., Two Corporate Drive, South San Francisco, California 94080, USA.
Amaral
M C
MC
Marshall
L
L
Huang
S G
SG
D052160:Benzothiazoles; D001681:Biological Assay / Q000379:methods*; D002232:Carbocyanines / Q000378:metabolism; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002463:Cell Membrane Permeability; D004140:Dinitrophenols / Q000494:pharmacology; D005954:Glucosephosphate Dehydrogenase / Q000378:metabolism; D007454:Iodides / Q000378:metabolism; D007473:Ion Channels; D007476:Ionophores / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D024101:Mitochondrial Proteins; D013050:Spectrometry, Fluorescence; D014475:Uncoupling Agents / Q000494:pharmacology; D000071256:Uncoupling Protein 1; D014634:Valinomycin / Q000494:pharmacology; D015003:Yeasts / Q000166:cytology* / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*
D052160:Benzothiazoles; D002232:Carbocyanines; D002352:Carrier Proteins; D004140:Dinitrophenols; D007454:Iodides; D007473:Ion Channels; D007476:Ionophores; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D014475:Uncoupling Agents; D000071256:Uncoupling Protein 1; D014634:Valinomycin; C012944:3,3'-dipropyl-2,2'-thiadicarbocyanine; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D005954:Glucosephosphate Dehydrogenase