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Published online 26 July 2004. doi:10.1083/jcb.200402120
The Rockefeller University Press, 0021-9525 $8.00
JCB, Volume 166, Number 3, 337-345
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Article

 Monitoring disulfide bond formation in the eukaryotic cytosol

Henrik Østergaard1, Christine Tachibana1,2, and Jakob R. Winther1

1 Department of Physiology, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby, Denmark
2 Department of Biochemistry, University of Washington, Seattle, WA 98195

Address correspondence to Jakob R. Winther, Dept. of Physiology, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby, Denmark. Tel.: 45-3327-5282. Fax: 45-3327-4765. email: jrw{at}crc.dk

Glutathione is the most abundant low molecular weight thiol in the eukaryotic cytosol. The compartment-specific ratio and absolute concentrations of reduced and oxidized glutathione (GSH and GSSG, respectively) are, however, not easily determined. Here, we present a glutathione-specific green fluorescent protein–based redox probe termed redox sensitive YFP (rxYFP). Using yeast with genetically manipulated GSSG levels, we find that rxYFP equilibrates with the cytosolic glutathione redox buffer. Furthermore, in vivo and in vitro data show the equilibration to be catalyzed by glutaredoxins and that conditions of high intracellular GSSG confer to these a new role as dithiol oxidases. For the first time a genetically encoded probe is used to determine the redox potential specifically of cytosolic glutathione. We find it to be –289 mV, indicating that the glutathione redox status is highly reducing and corresponds to a cytosolic GSSG level in the low micromolar range. Even under these conditions a significant fraction of rxYFP is oxidized.

Key Words: green fluorescent protein; glutathione; glutaredoxin; redox; oxidation

H. Østergaard's present address is Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark

Abbreviations used in this paper: 4-DPS, 4,4'-dithiodipyridine; GSH, reduced glutathione; GSSG, oxidized glutathione; NEM, N-ethyl-maleimide; NPM, N-(1-pyrenyl)maleimide; rGrx1p, recombinant His-tagged yeast Grx1p; rxYFP, redox sensitive YFP; TCA, trichloroacetic.


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