Mitochondrial and Cytosolic Isoforms of Yeast Fumarase Are Derivatives of a Single Translation Product and Have Identical Amino Termini

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.

mentioning

confidence: 99%

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

Porras

Padilla

Krayl

et al. 2006

“…Strains harboring the appropriate plasmids were grown overnight at 30°C in synthetic depleted medium containing 0.67% (w/v) yeast nitrogen base without amino acids (Difco) and 2% glucose or galactose (w/v) supplemented with the appropriate amino acids (50 g/ml). Plasmids pFT2 (Fum-WT) and pFSE24 (⌬SP) are described elsewhere (1,14). Plasmids encoding six histidine-tagged versions of fumarase and derivatives are described elsewhere (14).…”

Section: Methodsmentioning

confidence: 99%

Folding of Fumarase during Mitochondrial Import Determines its Dual Targeting in Yeast

Sass¹,

Karniely²,

Pines

2003

Self Cite

We have previously proposed that a single translation product of the FUM1 gene encoding fumarase is distributed between the cytosol and mitochondria of Saccharomyces cerevisiae and that all fumarase translation products are targeted and processed in mitochondria before distribution. Thus, fumarase processed in mitochondria returns to the cytosol. In the current work, we (i) generated mutations throughout the coding sequence which resulted in fumarases with altered conformations that are targeted to mitochondria but have lost their ability to be distributed; (ii) showed by mass spectrometry that mature cytosolic and mitochondrial fumarase isoenzymes are identical; and (iii) showed that hsp70 chaperones in the cytosol (Ssa) and mitochondria (Ssc1) can affect fumarase distribution. The results are discussed in light of our model of targeting and distribution, which suggests that rapid folding of fumarase into an import-incompetent state provides the driving force for retrograde movement of the processed protein back to the cytosol through the translocation pore.

“…9). In this respect Gpd2p is reminiscent of yeast fumarase, which is encoded by a single gene (FUM1) that produces a major cytosolic and a minor mitochondrial form (52). The FUM1 translation product contains a cleavable targeting signal and both the cytosolic and mitochondrial product appears to be processed by mitochondria, whereupon the mature cytosolic form returns to the cytosol via release from the mitochondria (53).…”

Section: Gpd2p Is Distributed Between Cytosol and Mitochondria-mentioning

confidence: 99%

Distinct Intracellular Localization of Gpd1p and Gpd2p, the Two Yeast Isoforms of NAD+-dependent Glycerol-3-phosphate Dehydrogenase, Explains Their Different Contributions to Redox-driven Glycerol Production

Valadi

Granath

Gustafsson

et al. 2004

Duringanaerobiosis Saccharomyces cerevisiae strongly increases glycerol production to provide for non-respiratory oxidation of NADH to NAD ؉ . We here report that respiratory-deficient cells become strictly dependent on the Gpd2p isoform of the NAD ؉ -linked glycerol-3-phosphate dehydrogenase (Gpd). The growth inhibition of respiratory incompetent cox18⌬ cells lacking GPD2 is reversed by the addition of acetoin, an alternative sink for NADH oxidation. Growth is also restored by addition of lysine or glutamic acid/glutamine, the synthesis of which involves production of mitochondrial NADH. Lysine produced a stronger growth stimulating effect than glutamic acid consistent with an upregulated expression of the IDP3 gene for peroxisomal synthesis of the glutamate precursor ␣-ketoglutarate. Gpd2p is known to be a cytosolic protein but possesses a classical mitochondrial presequence, which we show is sufficient for mitochondrial targeting. A partial mitochondrial localization of Gpd2p will provide for establishment of intramitochondrial redox balance under non-respiratory conditions. Gpd1p, the other Gpd isoform, is partly cytosolic and partly peroxisomal and becomes more strictly peroxisomal in respiratory-deficient mutants. The different cellular distribution of Gpd1p and Gpd2p thus appears to be the main reason Gpd1p cannot substitute for Gpd2p in cox18⌬gpd2⌬ cells, despite similar kinetic characteristics of the two iso-enzymes.