Is protein disulfide isomerase a redox-dependent molecular chaperone?

Abstract

Protein disulfide isomerase (PDI) is a multifunctional protein catalysing the formation of disulfide bonds, acting as a molecular chaperone and being a component of the enzymes prolyl 4-hydroxylase (P4H) and microsomal triglyceride transfer protein. The role of PDI as a molecular chaperone or polypeptide-binding protein is mediated primarily through an interaction of substrates with its b' domain. It has been suggested that this binding is regulated by the redox state of PDI, with association requiring the presence of glutathione, and dissociation the presence of glutathione disulfide. To determine whether this is the case, we investigated the ability of PDI to bind to a folding polypeptide chain within a functionally intact endoplasmic reticulum and to be dissociated from the alpha-subunit of P4H in vitro in the presence of reducing or oxidizing agents. Our results clearly demonstrate that binding of PDI to these polypeptides is not regulated by its redox state. We also demonstrate that the dissociation of PDI from substrates observed in the presence of glutathione disulfide can be explained by competition for the peptide-binding site on PDI.

Fig. 1. Synthesis of α2(I) C-propeptides in the presence of SP cells. RNA coding for either the wild-type or S3 C-propeptide was translated in a rabbit reticulocyte lysate in the presence of semi-permeabilized HT1080 cells (SP cells). SP cells were isolated by centrifugation. (A) Products of translation were immunoprecipitated using anti-HA antibody, separated by SDS–PAGE on a 10% polyacrylamide gel under reducing (lanes 1 and 2) or non-reducing conditions (lanes 3 and 4) and visualized by autoradiography. (B) Products of translation were left untreated (lanes 1 and 5) or chemically cross-linked with BMH (lanes 2–4 and 6–8), and immunoprecipitated with anti-HA, PDI or myc antibody. Precipitated samples were separated by SDS–PAGE on a 10% poly acrylamide gel under reducing conditions and visualized by autoradiography.

Fig. 2. The α2(I) C-propeptides can be cross-linked to PDI in a non-thiol-dependent manner. RNA coding for the α2(I), the α1(III) and the S3 C-propeptides were translated in a rabbit reticulocyte lysate in the presence of SP cells. SP cells were isolated by centrifugation and translation products were left untreated (lanes 1, 3 and 5) or chemically cross-linked with DSP (lanes 2, 4 and 6). Cross-linked samples were immunoprecipitated using the indicated antibodies before separation by SDS–PAGE on a 10% polyacrylamide gel, and were visualized by autoradiography.

Fig. 3. Determination of the redox state of PDI. (A) HT1080 cells were incubated for 20 min at room temperature alone (lanes 1 and 2), with DTT (lanes 3 and 4) or with DPS (lanes 5 and 6) before lysis in TCA. Free protein thiols were modified with AMS (lanes 2, 4 and 6) or not (lanes 1, 3 and 5), as described in Materials and methods. Proteins were resolved by SDS–PAGE on a 10% polyacrylamide gel, and PDI was detected by western blotting. (B) The HT1080 cell lysate was incubated for 20 min at room temperature alone (lane 1), with DPS (lane 2) or with DTT (lane 3). Samples were treated with NEM and separated by native gel electrophoresis through a 7.5% polyacrylamide gel. Proteins were transferred to nitrocellulose and PDI was detected by western blotting.

Fig. 4. Reduced and oxidized PDI associates with the S3 C-propeptide. The S3 C-propeptide was translated in a rabbit reticulocyte lysate in the presence of SP cells. SP cells were isolated by centrifugation, and then treated with DTT (lanes 1–4) or DPS (lanes 5–8). Excess DTT and DPS was removed by centrifugation before samples were chemi cally cross-linked with DSP or left untreated. Cross-linked and non-cross-linked samples were immunoprecipitated using the indicated antibodies before separation by SDS–PAGE on a 10% polyacrylamide gel, and translation products were visualized by autoradiography.

Fig. 5. PDI is oxidized when present as a subunit of the P4H complex. P4H was affinity purified by binding to poly-l-proline–Sepharose beads. Purified P4H was treated with 2 M urea, and the resulting dissociated PDI was treated with DPS (lane 5), DTT (lane 6) or left untreated. Similarly, intact HT1080 cells were treated with DPS (lane 2), DTT (lane 3) or left untreated. All samples were treated with NEM to prevent further disulfide exchange, and intact HT1080 cells were lysed. Proteins were separated by native gel electrophoresis on a 7.5% poly acrylamide gel, transferred to nitrocellulose, and PDI was detected using an anti-PDI antibody.

Fig. 6. P4H is not dissociated in the presence of an oxidizing agent but dissociates in the presence of a reducing agent. P4H was affinity purified by binding to poly-l-proline–Sepharose beads, and eluted with excess poly-l-proline. (A) Released P4H was left untreated (lane 1), or treated with 10 or 100 mM DTT (lanes 2 and 3) or DPS (lane 4). Proteins were separated by native gel electrophoresis on a 7.5% poly acrylamide gel, transferred to nitrocellulose, and PDI was detected using an anti-PDI antibody. (B) Released P4H was treated with DTT (lane 1) or DPS (lane 2), and proteins were separated by SDS–PAGE on a 10% polyacrylamide gel, transferred to nitrocellulose, and PDI was detected using an anti-PDI antibody.

Fig. 7. Dissociation of P4H in the presence of GSSG or mastoparan. P4H was affinity purified by binding to poly-l-proline–Sepharose beads, and eluted with excess poly-l-proline. Released P4H was left untreated (lane 1), treated with increasing concentrations of (A) glutathione disulfide or (B) mastoparan (lanes 2–4), or treated with DTT (lane 5). Samples were separated by native gel electrophoresis on a 7.5% polyacrylamide gel, transferred to nitrocellulose and PDI was detected using an anti-PDI antibody.