TY - JOUR

T1 - Monitoring disulfide bond formation in the eukaryotic cytosol

AU - Østergaard, Henrik

AU - Tachibana, Christine

AU - Winther, Jakob R.

PY - 2004

Y1 - 2004

N2 - 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.

AB - 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.

KW - Cystine

KW - Cytosol

KW - Disulfides

KW - Genes, Reporter

KW - Glutaredoxins

KW - Glutathione

KW - Oxidation-Reduction

KW - Oxidoreductases

KW - Proteins

KW - Saccharomyces cerevisiae

U2 - 10.1083/jcb.200402120

DO - 10.1083/jcb.200402120

M3 - Journal article

C2 - 15277542

VL - 166

SP - 337

EP - 345

JO - Journal of Cell Biology

JF - Journal of Cell Biology

SN - 0021-9525

IS - 3

ER -