Protein disulfide isomerase-like proteins play opposing roles during retrotranslocation

Abstract

Misfolded proteins in the endoplasmic reticulum (ER) are retained in the organelle or retrotranslocated to the cytosol for proteasomal degradation. ER chaperones that guide these opposing processes are largely unknown. We developed a semipermeabilized cell system to study the retrotranslocation of cholera toxin (CT), a toxic agent that crosses the ER membrane to reach the cytosol during intoxication. We found that protein disulfide isomerase (PDI) facilitates CT retrotranslocation, whereas ERp72, a PDI-like protein, mediates its ER retention. In vitro analysis revealed that PDI and ERp72 alter CT's conformation in a manner consistent with their roles in retrotranslocation and ER retention. Moreover, we found that PDI's and ERp72's opposing functions operate on endogenous ER misfolded proteins. Thus, our data identify PDI family proteins that play opposing roles in ER quality control and establish an assay to further delineate the mechanism of CT retrotranslocation.

Figure 1.

Retrotranslocation of the CTA1 subunit. (A) HeLa cells were incubated with CT for 90 min at 4 or 37°C with or without BFA or NEM. Cells were permeabilized, centrifuged, and the supernatant and pellet fractions were separated, subjected to nonreducing SDS-PAGE, and immunoblotted with the indicated antibodies. CTA, CTA1, and CTB are 28, 22, and 11 kD, respectively. (B) As in A except a CT mutant, R192H, was used. PDI is 58 kD. (C) As in A except where indicated, cells were treated with MG132. Polyubiquitinated proteins range from 100 to 300 kD. White lines indicate that intervening lanes have been spliced out.

Figure 2.

Down-regulation of PDI-like proteins. (A) Structural organization of PDI, ERp72, and ERp57. (B) PDI, ERp72, ERp57, and ERp29 protein levels were examined in wild-type (WT), PDI⁻, ERp72⁻, and ERp57⁻ cells. (C) Wild-type, PDI⁻, ERp72⁻, and ERp57⁻ cells were incubated with 2 μg/ml tunicamycin (TM) for 8 h, and BiP expression was assessed by SDS-PAGE and immunoblot analysis. BiP is 78 kD.

Figure 3.

PDI and ERp72 exert opposing effects on CT retrotranslocation. (A, top) Wild-type (WT), PDI⁻, ERp72⁻, and ERp57⁻ cells were incubated with CT for 45 or 90 min, permeabilized, and the supernatant fraction was analyzed as in Fig. 1. (bottom) The intensities of the CTA and CTA1 bands in the supernatant were quantified. Graphs show the mean ± SD (error bars) of two to four experiments. (B) Wild-type, PDI⁻, and ERp72⁻ cells were incubated with CT-GS, and the cell lysate was subjected to SDS-PAGE followed by immunoblotting with an anti-CTB antibody. Nonglycosylated and glycosylated CTB are 17 and 21 kD, respectively. (C) Whole cell lysates from CT-intoxicated wild-type, PDI⁻, and ERp72⁻ cells were prepared in the presence of NEM and subjected to immunoblotting with CTA antibody. (D) Wild-type, PDI⁻, and ERp72⁻ cells were incubated with CT for 45 or 90 min, and the cAMP level was measured by a cAMP Biotrak Enzyme Immunoassay System (GE Healthcare). Means ± SD of two to four experiments are shown. White lines indicate that intervening lanes have been spliced out.

Figure 4.

Opposing effects of PDI and ERp72 on the conformation of CT. (A) His-tagged ERp72 protein was purified from bacteria and analyzed by SDS-PAGE followed by Coomassie staining or immunoblotting with an antibody against ERp72. (B) CTA was incubated with BSA, PDI, or ERp72 followed by the addition of trypsin. Samples were subjected to SDS-PAGE followed by immunoblotting with an anti-CTA antibody.

Figure 5.

A general role of PDI and ERp72 in the retrotranslocation of ER misfolded proteins. (A, top) Wild-type (WT), PDI⁻, or ERp72⁻ cells were treated with MG132, and the cell lysate was subjected to SDS-PAGE followed by immunoblotting with the indicated antibodies. (bottom) The total ubiquitin signal intensity was measured. Graphs show the mean ± SD (error bars) of three to five experiments. (B) Wild-type, PDI⁻, or ERp72⁻ cells were treated with TNFα, and the lysate was subjected to SDS-PAGE followed by immunoblotting with an antibody against IκBα. IκBα is 39 kD. (C) CHO cells stably expressing cogTg (CHO-P) and PDI (CHO-PDI) or ERp72 (CHO-ERp72) were pulse labeled with [³⁵S]methionine and chased for the times indicated. CogTg was immunoprecipitated from the cell lysate with anti-Tg antibody and analyzed by SDS-PAGE. Data show quantification of the radioactive cogTg band intensity. (D) Diagram depicting the similarity between the quality control system in the ER and cytosol. See last paragraph of Results and discussion. White lines indicate that intervening lanes have been spliced out.