Novel Thioredoxin-related Transmembrane Protein TMX4 Has Reductase Activity

Yoshimi Sugiura,Kazutaka Araki,Shun-Ichiro Iemura,Tohru Natsume,Jun Hoseki,Kazuhiro Nagata

doi:10.1074/jbc.m109.082545

Abstract

In the endoplasmic reticulum (ER), a number of thioredoxin (Trx) superfamily proteins are present to enable correct disulfide bond formation of secretory and membrane proteins via Trx-like domains. Here, we identified a novel transmembrane Trx-like protein 4 (TMX4), in the ER of mammalian cells. TMX4, a type I transmembrane protein, was localized to the ER and possessed a Trx-like domain that faced the ER lumen. A maleimide alkylation assay showed that a catalytic CXXC motif in the TMX4 Trx-like domain underwent changes in its redox state depending on cellular redox conditions, and, in the normal state, most of the endogenous TMX4 existed in the oxidized form. Using a purified recombinant protein containing the Trx-like domain of TMX4 (TMX4-Trx), we confirmed that this domain had reductase activity in vitro. The redox potential of this domain (-171.5 mV; 30 degrees C at pH 7.0) indicated that TMX4 could work as a reductase in the environment of the ER. TMX4 had no effect on the acceleration of ER-associated degradation. Because TMX4 interacted with calnexin and ERp57 by co-immunoprecipitation assay, the role of TMX4 may be to enable protein folding in cooperation with these proteins consisting of folding complex in the ER.

Highlights

EXPERIMENTAL PROCEDURESCells and Antibodies—HeLa, human embryonic kidney (HEK293), and HepG2 cells were cultured in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum and antibiotics (supplemented with nonessential amino acids for HepG2 cells)
Disulfide bond formation is a rate-limiting step in the correct folding of nascent polypeptides of many secretory and membrane proteins
Because mRNA of BiP was induced by the same treatment, we concluded that TMX4 is a protein that does not respond to endoplasmic reticulum (ER) stress (Fig. 1, A and B)

Summary

EXPERIMENTAL PROCEDURES

Cells and Antibodies—HeLa, human embryonic kidney (HEK293), and HepG2 cells were cultured in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum and antibiotics (supplemented with nonessential amino acids for HepG2 cells). To remove the trigger factor portion of TF-TMX4-Trx, the eluted fraction was treated with HRV3C protease (Novagen) using the buffer conditions described in manufacturer’s instructions for 16 h at 4 °C. TMX4-Trx (1 ␮M) was incubated with 0.1 mM GSSG and various concentrations of GSH at 30 °C for 1 h in 0.1 M sodium phosphate buffer (pH 7.0) containing 1 mM EDTA and 1 mM NDSB-201 with ultracentrifugation at 120,000 ϫ g to remove aggregates of TMX4-Trx. After incubation, trichloroacetic acid (10%) was added to prevent further thiol-disulfide exchange. The precipitated pellet was solubilized in 0.1 M sodium phosphate buffer (pH 7.0), containing 2% SDS and 1 mM 4-acetamido-4Ј-maleidylstilbene-2,2Ј-disulfonic acid (AMS; Invitrogen) or 1 mM methoxypolyethylene glycol (average molecular weight 2000)-maleimide (mPEG2Kmal) (Sunbright ME-020MA; NOF Corporation, Japan), followed by incubation at 25 °C for 1 h to alkylate free sulfhydryl groups of cysteines. Fractions (250 ␮l each) were collected from the top, separated by SDS-PAGE, and examined by Western blot analysis

RESULTS

The noncatalytic cysteines were stable even when cells were

DISCUSSION