title: Characterization of alternative cytosolic forms and cellular targets of mouse mitochondrial thioredoxin reductase.
abstract: Thioredoxin reductase (TR) and thioredoxin (Trx) define a major cellular redox system that maintains cysteine residues in numerous proteins in the reduced state. Both cytosolic (TR1 and Trx1) and mitochondrial (TR3 and Trx2) enzymes are essential in mammals, but the function of the mitochondrial system is less understood. In this study, we characterized subcellular localization of three TR3 forms that are generated by alternative first exon splicing and that differ in their N-terminal sequences. Only one of these forms resides in mitochondria, whereas the two other isoforms are cytosolic. Consistent with this finding, TR3 did not have catalytic preferences for mitochondrial Trx2 versus cytosolic Trx1, both of which could serve as TR3 substrates. Similarly, TR1 was equally active with Trx1, Trx2, or a bacterial Trx. We generated recombinant selenoprotein forms of TR1 and TR3 and found that these enzymes were inhibited by zinc, but not by calcium or cobalt ions. We further developed a proteomic method for identification of targets of TRs in mammalian cells utilizing affinity columns containing recombinant TR3 forms differing in C-terminal sequences. Using this procedure, we found that Trx1 was the major target of TR3 in both rat and mouse liver cytosol. The truncated form of TR3 lacking selenocysteine was particularly efficient in binding Trx1, consistent with the previously observed role of truncated TR1 in apoptosis. Overall, these data establish that the function of TR3 is not limited to its role in Trx2 reduction.
authors.affiliation: Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 68588-0664, USA.
authors.forename: Anton A
authors.initials: AA
authors.lastname: Turanov
authors.forename: Dan
authors.initials: D
authors.lastname: Su
authors.forename: Vadim N
authors.initials: VN
authors.lastname: Gladyshev
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003600:Cytosol / Q000378:metabolism*; D006801:Humans; D008099:Liver / Q000378:metabolism; D051379:Mice; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D040901:Proteomics / Q000379:methods; D051381:Rats; D013880:Thioredoxin-Disulfide Reductase / Q000737:chemistry / Q000502:physiology*; D015032:Zinc / Q000737:chemistry
chemical_list: D013880:Thioredoxin-Disulfide Reductase; D015032:Zinc