Molecular cloning and characterization of a mitochondrial selenocysteine-containing thioredoxin reductase from rat liver.
A thioredoxin reductase (TrxR), named here TrxR2, that did not react with antibodies to the previously identified TrxR (now named TrxR1) was purified from rat liver. Like TrxR1, TrxR2 was a dimeric enzyme containing selenocysteine (Secys) as the COOH-terminal penultimate residue. A cDNA encoding TrxR2 was cloned from rat liver; the open reading frame predicts a polypeptide of 526 amino acids with a COOH-terminal Gly-Cys-Secys-Gly motif provided that an in-frame TGA codon encodes Secys. The 3'-untranslated region of the cDNA contains a canonical Secys insertion sequence element. The deduced amino acid sequence of TrxR2 shows 54% identity to that of TrxR1 and contained 36 additional residues upstream of the experimentally determined NH2-terminal sequence. The sequence of this 36-residue region is typical of that of a mitochondrial leader peptide. Immunoblot analysis confirmed that TrxR2 is localized almost exclusively in mitochondria, whereas TrxR1 is a cytosolic protein. Unlike TrxR1, which was expressed at a level of 0.6 to 1.6 microgram/milligram of total soluble protein in all rat tissues examined, TrxR2 was relatively abundant (0.3 to 0.6 microgram/mg) only in liver, kidney, adrenal gland, and heart. The specific localization of TrxR2 in mitochondria, together with the previous identification of mitochondria-specific thioredoxin and thioredoxin-dependent peroxidase, suggest that these three proteins provide a primary line of defense against H2O2 produced by the mitochondrial respiratory chain.
Lee
S R
SR
Laboratory of Cell Signaling, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Kim
J R
JR
Kwon
K S
KS
Yoon
H W
HW
Levine
R L
RL
Ginsburg
A
A
Rhee
S G
SG
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D018076:DNA, Complementary; D008930:Mitochondria, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D051381:Rats; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013347:Subcellular Fractions / Q000201:enzymology; D013880:Thioredoxin-Disulfide Reductase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism