Monitoring disulfide bond formation in the eukaryotic cytosol

Østergaard, Henrik; Tachibana, Christine; Winther, Jakob R.

doi:10.1083/jcb.200402120

Cited by 333Redox biology and oxidative stressSulfur Compounds in BiologyAdvanced Fluorescence Microscopy Techniques

Monitoring disulfide bond formation in the eukaryotic cytosol

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Østergaard, Henrik, Tachibana, Christine, Winther, Jakob R.·2004·The Journal of Cell Biology

DOI 10.1083/jcb.200402120PMC PMC2172265

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Abstract

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.

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Protein-based redox probes have drawbacks due, in part, to their reliance on cellular Grxs and their lag-time to equilibrate with cellular GSSG.
“Despite the advantage of the real-time values that can be tracked by these intracellular sensors, there may be other drawbacks of these protein-based probes due, in part, on their reliance on cellular Grxs and their lag-time to equilibrate with cellular GSSG (178, 319).”
From Redox regulation of mitochondrial function (2012) · cites this paper as [319]
In yeast cells, a redox-sensitive yellow fluorescent protein (rxYFP) forms disulfide bonds in proportion to intracellular GSH redox potential.
“In yeast cells, this has been accomplished by using a redox-sensitive yellow fluorescent protein (rxYFP) that forms disulfide bonds in proportion to intracellular GSH redox potential (319).”
From Redox regulation of mitochondrial function (2012) · cites this paper as [319]
Equilibration of the rxYFP sensor with intracellular GSH:GSSG relies on intracellular glutaredoxin.
“Using a series of genetically deficient yeast lines, it was shown that equilibration of the sensor with intracellular GSH:GSSG relies on intracellular Grx.”
From Redox regulation of mitochondrial function (2012) · cites this paper as [319]

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Authors: Østergaard, H., Tachibana, C., & Winther, J. R.
Affiliations: Carlsberg Laboratory (DK)
University of Washington (US)
ORCID: Winther, Jakob R.
Venue: The Journal of Cell Biology
Publisher: Rockefeller University Press
Published: 2004-07-26
Volume: 166
Issue: 3
Pages: 337-345
ISSN: 0021-9525 (ISSN-L)
1540-8140
DOI: 10.1083/jcb.200402120
PMC: PMC2172265
OpenAlex: W1998049513
Cited by: 333
References: 49
Citations by year: 2026: 6 · 2025: 12 · 2024: 16 · 2023: 8 · 2022: 10 · 2021: 11 · 2020: 10 · 2019: 17 · 2018: 16 · 2017: 19 · 2016: 15 · 2015: 17 · 2014: 22 · 2013: 16 · 2012: 22
FWCI: 4.03
Type: journal article
Language: en
Open access: green
www.ncbi.nlm.nih.gov
Licence: cc-by-nc-sa
Locations: The Journal of Cell Biology (publishedVersion)
PubMed Central (publishedVersion, cc-by-nc-sa)
PubMed (publishedVersion)
Topics: Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Redox biology and oxidative stress
Life Sciences › Biochemistry, Genetics and Molecular Biology › Biochemistry › Sulfur Compounds in Biology
Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Advanced Fluorescence Microscopy Techniques
Keywords: disulfide bond formation, glutathione, glutathione disulfide, glutaredoxin, yeast, thiol redox state
MeSH terms: Cystine, Cytosol, Disulfides, Glutathione, Oxidation-Reduction, Oxidoreductases, Proteins, Saccharomyces cerevisiae, Genes, Reporter, Glutaredoxins

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Monitoring disulfide bond formation in the eukaryotic cytosol

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