Identification and characterization of a novel thioredoxin-related transmembrane protein of the endoplasmic reticulum

The endoplasmic reticulum ( ER) contains a number of thiol- disulfide oxidoreductases of the protein- disulfide isomerase ( PDI) family that catalyze the formation of disulfide bonds in newly synthesized proteins. Here we describe the identification and characterization of a novel member of the human PDI family, TMX3 ( thioredoxinrelated transmembrane protein 3). The TMX3 gene encodes a protein of 454 amino acid residues that contains a predicted N- terminal signal sequence, a single domain with sequence similarity to thioredoxin and a CGHC active site sequence, a potential transmembrane domain, and a C- terminal KKKD tetrapeptide sequence that matches the classical KKXX- type consensus sequence for ER retrieval of type I transmembrane proteins. Endogenous TMX3 contains endoglycosidase Hsensitive glycans, localizes to the ER by immunofluorescence microscopy, and is present in the membrane fraction after alkaline extraction of the ER luminal content. The TMX3 transcript is found in a variety of tissues and is not up- regulated by the unfolded protein response. Circular dichroism spectroscopy of the recombinantly expressed luminal domain of TMX3 showed features typical of a properly folded protein of the alpha/beta type. The redox potential of recombinant luminal TMX3 was determined to - 0.157 V, similar to the values previously found for PDI and ERp57. Interestingly, TMX3 showed oxidase activity, and in human tissue- culture cells the protein was found partially in the oxidized form, potentially suggesting a function of the protein as a dithiol oxidase.