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Characterization of Glutathione Flux Between Subcellular Compartments
Author(s) -
Outten Caryn E,
Darch Maxwell,
McGee Crystal
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.773.8
Subject(s) - glutathione , cytosol , glutathione disulfide , redox , mitochondrial matrix , mitochondrion , biochemistry , chemistry , microbiology and biotechnology , flux (metallurgy) , oxidative phosphorylation , biophysics , biology , organic chemistry , enzyme
The redox‐active tripeptide glutathione (GSH) is critical for mitochondrial function since essential mitochondrial pathways such as Fe‐S cluster biogenesis, oxidative phosphorylation, and mitochondrial protein import are directly dependent on thiol‐disulfide balance. However, the fundamental mechanisms for controlling mitochondrial redox balance and regulating GSH:GSSG flux between subcellular compartments are not well characterized. To address these issues, we designed and implemented novel, genetically‐encoded green fluorescent protein (GFP)‐based redox sensors that report on the redox state of local GSH:GSSG pools in the intermembrane space (IMS) and matrix. By using these sensors combined with yeast genetics and cell biology techniques, we first demonstrated that the redox environment in the IMS is maintained separately from the matrix and cytosol (1, 2). In the current study, we have assessed the effects of altered GSH metabolism on IMS and matrix redox control and investigated the pathways for GSH:GSSG exchange between subcellular compartments. To study GSH:GSSG flux within the eukaryotic cell, we designed an experimental system using yeast strains that overexpress the GSH:GSSG plasma membrane importer, Hgt1, which leads to rapid intracellular accumulation of GSH or GSSG when added to the growth media. These studies show that the matrix GSH:GSSG pool is more insulated from redox perturbations and that vacuolar GSH:GSSG storage plays an important role in buffering both the cytosol and mitochondrial matrix from dramatic redox changes. Furthermore, our results demonstrate that GSH:GSSG flux has a direct impact on thiol‐disulfide balance in the IMS in vivo. Support or Funding Information This research is funded by the National Institute of General Medical Sciences grant number R35 GM118164 to Caryn E. Outten.Using GFP‐based redox sensors to monitor subcellular GSH:GSSG redox changes in yeast