Novel thioredoxin-related transmembrane protein TMX4 has reductase activity

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.

FIGURE 1.

Expression profile of TMX4. A, expression of TMX4 mRNA before and after ER stress. Total mRNA from HeLa cells that were not treated (NT) or were treated with 300 nm thapsigargin (Tg), 2 μg/ml tunicamycin (Tm), and 5 mm DTT for 6 h were analyzed by Northern blotting. BiP was used as a positive control for induction by ER stress, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was an internal control. B, quantification of TMX4 mRNA before and after ER stress. Northern blot signals were quantified, as in A, and normalized to GAPDH mRNA levels. The results are means ± S.D. of three independent experiments. C, tissue distribution of mRNA expression of TMX4. A multiple human tissue blot was hybridized with TMX4 C-terminal cDNA (660–1050) and β-actin cDNA as a control.

FIGURE 2.

Subcellular localization of TMX4. A, domain structure of human TMX4. TMX4 has an ER targeting signal sequence, one Trx-like domain with a CXXC motif (CPSC), and one transmembrane region. One N-glycosylation site is present in the Trx-like domain. B, immunofluorescence analysis of HeLa cells expressing TMX4-HA. Cells were co-stained with anti-HA (green) and anti-PDI (red, ER marker). C, cellular fractionation assay. Cells expressing TMX4-HA were lysed, and the nuclear fraction was removed by centrifugation. The PNS was fractionated by sequential centrifugation into cytosolic (Cy) and microsomal (Ms) fractions. The fractions were analyzed by Western blotting using anti-HA, anti-calnexin (CNX, an ER marker protein), and anti-tubulin (a cytosolic marker protein). D, Endo H digestion. Cell lysates expressing TMX4-HA were incubated with or without Endo H and analyzed by Western blotting using an HA antibody. E, alkaline extraction of microsomal TMX4. A microsomal fraction prepared from cells transiently expressing TMX4-HA was fractionated by ultracentrifugation after no treatment (lanes 1 and 2) or treatment with alkaline buffer (Na₂CO₃, lanes 3 and 4) or 1% Nonidet P-40 (lanes 5 and 6). The fractions were analyzed by Western blotting using anti-calnexin (an ER membrane marker protein) and anti-PDI (an ER luminal marker protein). F, trypsin digestion of TMX4. A PNS fraction prepared from cell lysates expressing TMX4-HA was treated with the indicated concentration of trypsin and analyzed by Western blotting using anti-HA; anti-calnexin N terminus, which recognizes the luminal portion of calnexin; anti-calnexin C terminus, which recognizes the cytosolic portion of calnexin; and anti-BiP (an ER luminal marker protein).

FIGURE 3.

In vivo redox states of TMX4. A, redox state of TMX4-HA. TMX4-HA transfected cells were lysed by addition of trichloroacetic acid to prevent further disulfide exchange. Trichloroacetic acid precipitates were resolved in 0.1 m sodium phosphate buffer (pH 7.0), 2% SDS, with or without 1 mm mPEG2K-mal. The mPEG2K-mal modifications were performed at 25 °C for 1 h and analyzed by Western blotting (WB). Reduced (Red) and oxidized (Ox) TMX4 were prepared by pretreatment of cells with DTT and H₂O₂. Two cysteine mutants of TMX4, TMX4-CXXC-3S and TMX4-SXXS-3C, in which cysteines outside the CXXC motif or in the CXXC motif were mutated to serines, respectively, were used to clarify which cysteines are crucial for the effect of DTT or H₂O₂ treatment. B, redox state of endogenous TMX4. The redox state of endogenous TMX4 was analyzed by AMS modification.

FIGURE 4.

In vitro analysis of the Trx-like domain of TMX4. A, insulin reduction assay of recombinant TMX4-WT-Trx and TMX4-SS-Trx. Enzyme-catalyzed reduction of insulin disulfide bonds by GSH is coupled to the reduction of GSSG by glutathione reductase. The insulin reductase activity of the enzyme was measured with 8 mm GSH at 25 °C by spectrophotometrically monitoring NADPH consumption, which is concomitant with GSSG reduction by glutathione reductase. CBB, Coomassie Brilliant Blue. Red, reduced; Ox, oxidized. B, upper, redox equilibrium assay with glutathione at 30 °C. The free sulfhydryl groups of the cysteine residues were modified with AMS after incubation with different [GSH]²/[GSSG] ratios. Lower, measured redox equilibrium constant of TMX4. The apparent equilibrium constant between TMX4 and glutathione was determined by the nonlinear least square fitting of the data in the upper panel.

FIGURE 5.

ER protein complexes containing TMX4. A, fractionation of endogenous TMX4 by sucrose density gradient centrifugation. HEK293 cells were lysed with 3% digitonin and fractionated using a 10–40% sucrose density gradient. The fractions were separated by SDS-PAGE and analyzed by Western blotting with the indicated antibodies. B, proteome analysis of TMX4-interacting proteins. TMX4-FLAG (bait)-overexpressed HEK293 cells were lysed mechanically. Cell lysate was subjected to immunoprecipitation with anti-FLAG antibody followed by liquid chromatography/mass spectrometry analysis. Average number of detected peptides derived from each protein in four experiments was shown. WT, wild type. C, co-immunoprecipitation (IP) of TMX4 with calnexin (CNX). HEK293 cells overexpressing TMX4-FLAG and calnexin-HA were labeled for 16 h with [³⁵S]methionine/cysteine at 24 h after transfection. Cells were lysed with 3% digitonin and subjected to immunoprecipitation. Catalytic cysteine mutant of TMX4 (TMX4-AA) was used to check whether interaction between TMX4 and calnexin was independent of disulfide bond formation via CXXC motif or not. Arrowhead indicates endogenous calnexin. This band was confirmed to correspond to the endogenous calnexin. D, co-immunoprecipitation of TMX4 with ERp57. HEK293 cells overexpressing TMX4-HA and ERp57-FLAG were labeled for 1 h with [³⁵S]methionine/cysteine. Cells were lysed with 1% Nonidet P-40 and subjected to immunoprecipitation. Catalytic cysteine mutants of TMX4 (TMX4-CS and TMX4-SS) were used to see whether this interaction was dependent of covalent binding via its catalytic cysteines or not. Asterisk shows nonspecific band.