WRB shows sequence similarity to yeast Get1. (A) Multiple sequence alignment of Get1 homologs of eukaryotic organisms. Identical residues are indicated in white on a red background and similar residues in red. Alignment was performed by ESPript software (Gouet et al., 1999). (B) Linear outline of WRB. The predicted TMDs (red bars), the coiled-coil (cc) domain and the peptides (P1 and P2) used to raise anti-WRB antibodies are indicated. (C) Predicted membrane topology of WRB.

TRC40 associates with WRB–HA at the ER. (A) Co-immunoprecipitation of WRB–HA with TRC40 (left panel) and TRC40 with WRB–HA (right panel). HeLa cells were transfected with WRB–HA alone and with WRB–HA and TRC40 (left panel), or with TRC40 alone and with WRB–HA and TRC40 (right panel). 20% of the cell lysates were directly loaded onto the gel (lanes 1, 5 and 9, 13) and the rest were used for immunoprecipitation with the anti-TRC40 antibody (lanes 3 and 7), the anti-HA antibody (lanes 11, 15), an unrelated antibody (anti-myc) (lanes 4, 8 and 12, 16) or without an antibody (lanes 2, 6 and 10, 14). Lysates (TOT) and immunoprecipitates (IP) were analyzed by SDS-PAGE and immunoblotting using the anti-HA (left panel) or anti-TRC40 (right panel) antibodies. The asterisk indicates light chains of anti-myc antibody. (B) Colocalization of YFP–TRC40 with WRB–CFP at the ER. WRB–CFP was co-expressed with YFP–TRC40 (upper panel) or with YFP (middle panel), or YFP–TRC40 was expressed alone (lower panel). Cells were permeabilized with digitonin to release cytosolic content, and the CFP (red), YFP (green) signals or both signals were recorded over time using confocal microscopy. Untreated cells or cells treated with digitonin for 40 seconds and 400 seconds are shown. Scale bars: 10 μm. A graphical interpretation of the colocalization of YFP–TRC40 (green) with WRB–CFP (red) is shown on top of the figure.

Recombinant WRBcc specifically prevents TRC40-dependent membrane insertion of TA proteins. (A) WRBcc was expressed in E. coli and tested for its ability to affect TRC40-mediated membrane insertion (glycosylation) of the TA protein RAMP4op. HZZ-RAMP4op–MBP–TRC40 complexes were produced in E. coli, purified and incubated in the absence (lane 1) or presence (lanes 2–7) of RMs, and increasing amounts of purified WRBcc (lanes 3–6) or MBP (lane 7). Proteins were analyzed by SDS-PAGE and immunoblotting using anti-opsin, anti-MBP or anti-WRB antibodies. Quantification of relative amounts of glycosylated (membrane-inserted) HZZ-RAMP4op is shown in the graph. Error bars ± s.d. (B) Effect of WRBcc on membrane insertion of cytochrome b5 (Cb5) co-purified with MBP–TRC40. HZZ-Cb5op–MBP–TRC40 complexes were incubated in the absence (lane 1) or presence (lanes 2–7) of RMs, and increasing amounts of purified WRBcc (lanes 3–6) or MBP (lane 7). Proteins were analyzed by SDS-PAGE and immunoblotting as described above. (C) Effect of WRBcc on membrane insertion of cytochrome b5 (Cb5). HZZ-Cb5op was incubated in the absence (lane 1) or presence (lanes 2–7) of RMs, and increasing amounts of purified WRBcc (lanes 3–6) or MBP (lane 7). Proteins were analyzed by SDS-PAGE and immunoblotting as described above.

WRB is a 19 kDa membrane protein of the ER. (A) Identification of WRB–HA and HA–WRB. N-terminally or C-terminally HA-tagged forms of WRB (HA–WRB or WRB–HA) were expressed in HeLa cells. Proteins were separated by SDS-PAGE and analyzed by immunoblotting using the pre-immune serum (lanes 1–3), the anti-WRB serum (lanes 4–6) or anti-HA antibody (lanes 7–9). The asterisks indicate incomplete WRB. (B) Membrane association of WRB–HA. Untransfected HeLa cells (lanes 1–3) or HeLa cells transfected with HA–WRB (lanes 4–6) were analyzed by SDS-PAGE and immunoblotting using anti-WRB, anti-TRAM and anti-GAPDH antibodies. HeLa cells were permeabilized with digitonin, and cell constituents separated into a cytosolic and membrane fractions by centrifugation. Proteins of whole cell lysate (lanes 1 and 4), membrane (lanes 2 and 5) and cytosolic fractions (lanes 3 and 6) were analyzed. (C) Localization of WRB–CFP. CFP-tagged WRB (WRB–CFP) or CFP were expressed in RPE-1 cells and visualized by fluorescence microscopy. (D) ER localization of WRB–HA. WRB–HA was expressed in RPE-1 cells, immunostained with an anti-HA antibody and visualized with a fluorescent secondary antibody (red) by fluorescence microscopy. As an ER marker, calreticulin was visualized similarly using an anti-calreticulin antibody and a fluorescent secondary antibody (green).

WRBcc interacts with TRC40. (A) Purified GST–TRC40 and MBP–WRBcc interact with each other. MBP (lane 1) and MBP–WRBcc (lane 3) were bound to amylose resin. GST–TRC40 (lane 2) was then incubated with the resins, and the unbound and bound proteins characterized by SDS-PAGE and Coomassie Blue staining. (B) HisWRBcc co-immunoprecipitates with TRC40 and TRC40 with HisWRBcc. HisWRBcc (lanes 1–4) and TRC40 (lanes 9–12) were expressed alone or together (lanes 5–8 and 13–16) in HeLa cells, and either TRC40 (left panel) or hisWRBcc (right panel) was immunoprecipitated by their respective antibodies (anti-TRC40 or anti-his). Co-immunoprecipitated proteins were then characterized by western blot analysis using the anti-his (left panel) or anti-TRC40 (right panel) antibody.