Partition and Turnover of Glutathione Reductase from Saccharomyces cerevisiae: A Proteomic Approach

Glutathione reductase (Glr1) is a low abundance protein involved in defense mechanisms against reactive oxygen species. Expressed on cytosolic ribosomes, the same gene, GLR1, uses alternative start codons to generate two forms of Glr1. Translation from the first AUG codon generates the mitochondrial...

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Bibliographic Details
Published in:Journal of proteome research 2013-06, Vol.12 (6), p.2885-2894
Main Authors: Couto, Narciso, Malys, Naglis, Gaskell, Simon J, Barber, Jill
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Base Sequence
Codon, Initiator
Cytosol - chemistry
Cytosol - enzymology
Gene Expression Regulation, Fungal
Isoenzymes - chemistry
Isoenzymes - genetics
Isoenzymes - metabolism
Mitochondria - chemistry
Mitochondria - enzymology
Molecular Sequence Data
Peptide Chain Initiation, Translational - genetics
Protein Structure, Tertiary
Proteomics
Saccharomyces cerevisiae - enzymology
Saccharomyces cerevisiae - genetics
Saccharomyces cerevisiae Proteins - chemistry
Saccharomyces cerevisiae Proteins - genetics
Saccharomyces cerevisiae Proteins - metabolism
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Summary:Glutathione reductase (Glr1) is a low abundance protein involved in defense mechanisms against reactive oxygen species. Expressed on cytosolic ribosomes, the same gene, GLR1, uses alternative start codons to generate two forms of Glr1. Translation from the first AUG codon generates the mitochondrial form incorporating a presequence necessary for import; translation from the second AUG codon yields the cytosolic counterpart. Proteomic strategies were used to analyze the N-terminal sequences and the turnover of Saccharomyces cerevisiae Glr1. The N-terminus of cytosolic Glr1 was found normally to be N-acetylserine. When a Glr1-overproducing strain was employed, unprocessed mitochondrial Glr-1 with N-terminal acetylmethionine also accumulated in the cytosol. The processed mitochondrial Glr1 was surprisingly found to have three alternative N-termini, none of them acetylated. Mitochondrial Glr1 was turned over faster than the cytosolic form by a factor of about 2, consistent with the importance of redox homeostasis in the mitochondria. These experiments also allowed us to estimate the extent of “leaky scanning” in the synthesis of Glr1. Surprisingly, the second AUG appears to be responsible for most of the cellular Glr1. This is the first report of protein turnover measurements of a low-abundance protein distributed in different compartments of a eukaryotic cell.
ISSN:1535-3893
1535-3907
DOI:10.1021/pr4001948