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

Abstract

Glutaredoxins belong to a family of small proteins with glutathione-dependent disulfide oxidoreductase activity involved in cellular defense against oxidative stress. The product of the yeast GRX2 gene is a protein that is localized both in the cytosol and mitochondria. To throw light onto the mechanism responsible for the dual subcellular distribution of Grx2 we analyzed mutant constructs containing different targeting information. By altering amino acid residues around the two in-frame translation initiation start sites of the GRX2 gene, we could demonstrate that the cytosolic isoform of Grx2 was synthesized from the second AUG, lacking an N-terminal extension. Translation from the first AUG resulted in a long isoform carrying a mitochondrial targeting presequence. The mitochondrial targeting properties of the presequence and the influence of the mature part of Grx2 were analyzed by the characterization of the import kinetics of specific fusion proteins. Import of the mitochondrial isoform is relatively inefficient and results in the accumulation of a substantial amount of unprocessed form in the mitochondrial outer membrane. Substitution of Met 35 , the second translation start site, to Val resulted in an exclusive targeting to the mitochondrial matrix. Our results show that a plethora of Grx2 subcellular localizations could spread its antioxidant functions all over the cell, but one single Ala to Gly mutation converts Grx2 into a typical protein of the mitochondrial matrix.

A trans -Dominant Form of Gag Restricts Ty1 Retrotransposition and Mediates Copy Number Control

“…This result raises the possibility that full-length Ty1 and Ty1i transcripts may utilize an internal ribosome entry site (65) upstream of AUG1 or AUG2 to drive synthesis of p22. Other mechanisms by which p22 could be translated from full-length Ty1 mRNA are leaky scanning, where scanning ribosomes sometimes initiate translation from an alternate AUG codon (66)(67)(68)(69) or translation reinitiation in which translation starts at a downstream AUG after translation of an ORF situated upstream (70,71). Although leaky scanning and translation reinitiation remain possible mechanisms, both require closely spaced AUGs.…”

Alternative translation start sites and hidden coding potential of eukaryotic mRNAs

“…Thus, alternative TISs are used to spread Grx2 antioxidant functions all over the yeast cell. (22) It was found that an alternative translation initiation was the major mechanism for the synthesis of membrane-bound and cytosolic forms of Debaryomyces hansenii 3 0 , 5 0 -bisphosphate nucleotidase also characterized by significant difference in substrate specificity. (23) Cytoplasmic and mitochondrial isoforms of Saccharomyces cerevisiae alanyl-tRNA synthetase and glycyl-tRNA synthetase are also translated from alternative TISs by leaky scanning mechanism.…”

Selenite-induced cell death in Saccharomyces cerevisiae: protective role of glutaredoxins

“…S. cerevisiae contains eight GRXs (Grx1p to Grx8p), which are in different cell compartments. The dithiol GRXs Grx1p and Grx2p (CPYC active site) are mainly cytosolic, but a minor portion of Grx2p is located within mitochondria (Luikenhuis et al, 1998;Porras et al, 2006). Despite the proposed role of Grx1p and Grx2p as general thiol oxidoreductases, their absence causes only moderate hypersensitivity to superoxide (in the case of Grx1p) and to superoxide and hydroperoxide (in the case of Grx2p) (Eckers et al, 2009;Luikenhuis et al, 1998).…”