Identification and Characterization of a Novel Thioredoxin-related Transmembrane Protein of the Endoplasmic Reticulum

Abstract

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 (thioredoxin-related 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 ␣/␤ 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.The formation of disulfide bonds plays a critical role for the correct folding of most secretory and plasma membrane proteins in the endoplasmic reticulum (ER).1 Whereas folding of certain proteins proceeds by the sequential formation of native disulfides, it is clear that other proteins (e.g. the low density lipoprotein receptor) (1) form long range nonnative disulfides that are later rearranged as an integral folding step in living cells. Both the formation and the rearrangement of disulfide bonds often rely on the catalysis by thiol-disulfide oxidoreductases.Protein-disulfide isomerase (PDI) is the founding member of a family of thiol-disulfide oxidoreductases in the ER (reviewed in Ref.2). The protein contains four domains, named a, b, b, and a, all with homology to thioredoxin. Like this cytosolic reductase, the catalytic a and a domains of PDI contain tetrapeptide CXXC active site sequences (where X denotes any amino acid), for catalysis of thiol-disulfide exchange reactions. These reactions proceed through transient mixed disulfide intermediates between enzyme and substrate and lead to the oxidation (formation), reduction (breaking), or isomerization (rearrangement) of substrate cysteines. The two active site cysteines in oxidoreductases are found either in the oxidized disulfide or the reduced dithiol form; the disulfide form determines the function of the enzyme as an oxidase, whereas the dithiol form allows it to act as a reductase and/or an isomerase. Of the noncatalytic...

The Human Selenoprotein VCP-interacting Membrane Protein (VIMP) Is Non-globular and Harbors a Reductase Function in an Intrinsically Disordered Region

“…Instead, we established the reduction potential for Cys174-Cys188 in cVIMP-Cys to provide an indication of the stability of the selenosulfide bond in the wild-type sequence. A priori, the method of choice for these studies was fluorescence spectroscopy, which we have previously used to determine the reduction potential for certain ER oxidoreductases (37,38). However, we observed no significant difference between oxidized and reduced cVIMP-Cys by this method (data not shown).…”

Endoplasmic reticulum: Reduced and oxidized glutathione revisited

“…This indicates that oxidation of ERp57a9 is specifically catalyzed and that, at variance with previous models (Appenzeller-Herzog, 2011), GSSG-driven oxidation of reduced PDIs is probably a minor pathway (at least in HeLa cells). Whether the oxidized fraction of ERp57a9 [and of other family members such as TMX3 (Haugstetter et al, 2005)] is maintained by Ero1 (Ramming and AppenzellerHerzog, 2012) or by other oxidative pathways (Ruddock, 2012) is presently unclear. Another interesting implication following the determination of E GSH (ER) relates to the question, how a catalytically active (i.e.…”

Limiting Role of Protein Disulfide Isomerase in the Expression of Collagen-tailed Acetylcholinesterase Forms in Muscle

“…These results indicate that thiol oxidoreductase activity is required for folding of catalytically active AChE molecules. However, all PDI family members bear thiol oxidoreductase activity, and skeletal muscle fibers appear to express at least three variants (37)(38)(39). To determine whether PDI was specifically required for ColQ-AChE expression, we transfected QMCs with a plasmid encoding a qPDI-shRNA to selectively knock down PDI expression without affecting other thiol oxidoreductases.…”