Subcellular localization of methionine sulphoxide reductase A (MsrA): evidence for mitochondrial and cytosolic isoforms in rat liver cells.
Proteins are sensitive to reactive oxygen species, and the accumulation of oxidized proteins has been implicated in the aging process and in other age-related pathologies. In proteins, methionine residues are especially sensitive to oxidation, leading to S - and R -methionine sulphoxide diastereoisomers, the reversion of which is achieved by the peptide methionine sulphoxide reductases MsrA and MsrB respectively. The MsrA enzyme, in addition to its role in repair, forms part of the reactive oxygen species scavenging systems that are important in cellular antioxidant defence. MsrA is present in most living organisms, and the mammalian enzyme has been detected in all tissues investigated. In the present study, we investigated the subcellular distribution of MsrA in rat liver cells. Since it seemed likely that MsrA may be localized in areas where reactive oxygen species are produced, rat liver mitochondrial matrix and cytosolic extracts were prepared. The presence of MsrA was assayed in these subcellular compartments by monitoring peptide methionine sulphoxide reductase enzymic activity, by Western blotting and by in situ immunolocalization by electron microscopy using a specific antibody. Moreover, MsrA was identified by MS in a partially purified cytosolic fraction and in a mitochondrial matrix crude extract. Rat MsrA isoforms are encoded by a single gene, and it is suggested that the precursor of the mitochondrial form contains an N-terminal cleavable signal sequence that localizes the MsrA to this organelle. Finally, two-dimensional gel electrophoresis followed by Western-blot analysis of partially purified MsrA from the cytosol and mitochondria, and comparison with the two-dimensional patterns of oxidized recombinant MsrA, revealed oxidative modifications of cysteine residues.
Vougier
Stéphanie
S
Laboratoire de Biologie et Biochimie Cellulaire du Vieillissement, Université Denis Diderot-Paris 7, CC 7128, 2 place Jussieu, 75251 Paris Cedex 05, France.
Mary
Jean
J
Friguet
Bertrand
B
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D003600:Cytosol / Q000201:enzymology*; D015180:Electrophoresis, Gel, Two-Dimensional; D007527:Isoenzymes; D008099:Liver / Q000201:enzymology*; D013058:Mass Spectrometry; D051150:Methionine Sulfoxide Reductases; D008854:Microscopy, Electron; D008930:Mitochondria, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions
D007527:Isoenzymes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D010088:Oxidoreductases; D051150:Methionine Sulfoxide Reductases; C027219:methionine sulfoxide reductase