Monitoring disulfide bond formation in the eukaryotic cytosol
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Monitoring disulfide bond formation in the eukaryotic cytosol. / Østergaard, Henrik; Tachibana, Christine; Winther, Jakob R.
I: Journal of Cell Biology, Bind 166, Nr. 3, 2004, s. 337-345.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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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 -
ID: 43973558