Two isoforms of Saccharomyces cerevisiae glutaredoxin 2 are expressed in vivo and localize to different subcellular compartments

Abstract

Glutaredoxin (Grx)2 from Saccharomyces cerevisiae is a member of the two-cysteine (dithiol) subfamily of Grxs involved in the defence against oxidative stress in yeast. Recombinant yeast Grx2p, expressed in Escherichia coli, behaves as a 'classical' Grx that efficiently catalyses the reduction of hydroxyethyl disulphide by GSH. Grx2p also catalyses the reduction of GSSG by dihydrolipoamide with even higher efficiency. Western blot analysis of S. cerevisiae crude extracts identifies two isoforms of Grx2p of 15.9 and 11.9 kDa respectively. The levels of these two isoforms reach a peak during the exponential phase of growth in normal yeast extract/peptone/dextrose ('YPD') medium, with the long form predominating over the short one. From immunochemical analysis of subcellular fractions, it is shown that both isoforms are present in mitochondria, but only the short one is detected in the cytosolic fraction. On the other hand, only the long form is prominent in microsomes. Mitochondrial isoforms should represent the processed and unprocessed products of an open reading frame (YDR513W), with a putative start codon 99 bp upstream of the GRX2 start codon described thus far. These results indicate that GRX2 contains two in-frame start codons, and that translation from the first AUG results in a product that is targeted to mitochondria. The cytosolic form would result either by initiation from the second AUG, or by differential processing of one single translation product.

One Single In-frame AUG Codon Is Responsible for a Diversity of Subcellular Localizations of Glutaredoxin 2 in Saccharomyces cerevisiae

“…It also attenuates apoptosis by preventing cytochrome c release in HeLa cells (31). In contrast to the mammalian protein, Grx2 from the yeast S. cerevisiae has a dual subcellular localization but originates from one single transcript (16). Only a very few other proteins with a similar behavior have been identified to date (29).…”

“…Only a very few other proteins with a similar behavior have been identified to date (29). The dual localization of yeast Grx2 is related to the presence of an in-frame translation start site (TSS) upstream of the canonical one, which produces a potential mitochondrial N-terminal targeting sequence (16). Spreading of the protein over subcellular compartments is apparently the consequence of post-transcriptional phenomena taking place during and/or after translation.…”

“…The functions of both systems overlap to a certain extent, although the major difference is that Grxs have a glutathione binding site and are capable of reducing GSH protein-mixed disulfides (6,13,14). Mitochondrial isoforms of each member of both systems have been characterized in yeast (15)(16)(17) and mammals (18 -20). Three Grx subfamilies have been distinguished (21).…”

“…These five Grxs also differ in regard to their subcellular localization. Grx1 is cytosolic, Grx3 and Grx4 are nuclear (17,23), Grx5 is mitochondrial (24), and Grx2 has a dual localization in the cytosol and mitochondria (16). Grx2 stands out among other Grxs for its efficiency in transferring reducing equivalents from reduced lipoamide to oxidized glutathione (25).…”

The Dithiol Glutaredoxins of African Trypanosomes Have Distinct Roles and Are Closely Linked to the Unique Trypanothione Metabolism

“…Their considerable variation in specificity allows them to participate in a large number of biological processes. All organisms have an individual set of isoforms that occur in the cytosol, mitochondria, or nucleus of different cells (1)(2)(3). Structurally, two main groups are distinguished, dithiol Grxs containing a CXXC active site sequence (mostly CPYC) and monothiol Grxs with a CXXS motif (mostly CGFS) (4).…”