Unfolded cholera toxin is transferred to the ER membrane and released from protein disulfide isomerase upon oxidation by Ero1 - PubMed (original) (raw)

Unfolded cholera toxin is transferred to the ER membrane and released from protein disulfide isomerase upon oxidation by Ero1

Billy Tsai et al. J Cell Biol. 2002.

Abstract

The toxic effect of cholera toxin (CT) on target cells is caused by its A1 chain. This polypeptide is released from the holotoxin and unfolded in the lumen of the ER by the action of protein disulfide isomerase (PDI), before being retrotranslocated into the cytosol. The polypeptide is initially unfolded by binding to the reduced form of PDI. We show that upon oxidation of the COOH-terminal disulfide bond in PDI by the enzyme Ero1, the A1 chain is released. Both yeast Ero1 and the mammalian Ero1alpha isoform are active in this reaction. Ero1 has a preference for the PDI-toxin complex. We further show that the complex is transferred to a protein at the lumenal side of the ER membrane, where the unfolded toxin is released from PDI by the action of Ero1. Taken together, our results identify Ero1 as the enzyme mediating the release of unfolded CT from PDI and characterize an additional step in retrotranslocation of the toxin.

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Figures

Figure 1.

Figure 1.

An ER activity induces release of the A1 chain from PDI. (A) Isolated A subunit (70 nM) was incubated with either BSA (3 μM), purified mammalian PDI (3 μM), or ER extract (3 mg/ml) in GSH (1 mM). Where indicated, trypsin (100 μg/ml) was added. Samples were analyzed by nonreducing SDS-PAGE and immunoblotting with a CT antibody. The positions of the proteolytically cleaved A subunit (containing A1 and A2 chains linked by a disulfide bond) and of the A1 chain are indicated. (B) Isolated A subunit (70 nM) was incubated with either BSA (3 μM), ER extract (3 mg/ml) or purified PDI (3 μM) in GSH (1 mM). A carbodiimide crosslinker (EDAC) was then added. Samples were analyzed as in (A). PDI-A represents a crosslinked product between PDI and the A subunit. * indicates an unidentified band. (C) Isolated A subunit (70 nM) was first incubated with purified PDI (3 μM) in GSH (1 mM), followed by addition of either BSA (3 μM) or ER extract (3 mg/ml). Samples were analyzed as in (B).

Figure 2.

Figure 2.

Ero1 induces the release of the A1 peptide from reduced PDI. (A) Protein from five equivalents of dog pancreatic microsomes were treated with or without endo H and analyzed by SDS-PAGE followed by immunoblotting with antibodies to Ero1α/β (lanes 1 and 2), Ero1α (lanes 3 and 4), or Ero1β (lanes 5 and 6). Purified mammalian Ero1α (1 μg), analyzed in the Coomassie gel (lane 7), was tested with antibodies to Ero1α/β, Ero1α, or Ero1β (lanes 8–10). x represents an uncharacterized glycosylated form of Ero1α. (B) ER extract or Ero1- depleted extract (depleted by incubation with either antibody 193 or 194) were analyzed by immunoblotting with antibodies to Ero1α/β (antibody 194, lanes 1–3), Ero1α (lanes 4–6), or Ero1β (lanes 7–9). Lanes 10–14 show the results of release assays. Isolated A subunit (70 nM) and PDI (3 μM) were incubated in GSH (1 mM) followed by addition of BSA (2 mg/ml), ER extract (2 mg/ml), Ero1-depleted extract (depleted by antibody 193 or 194), or GSSG (30 mM; lanes 10–14). The samples were analyzed as in Fig. 1 B. (C) Purified mammalian Ero1α (0.3 μM or 3 μM) was added to the PDI–toxin complex and analyzed as in Fig. 1 B (lanes 1–3). Purified yeast Ero1 (Coomassie gel, lane 4) was added (0.3 μM or 1.5 μM) to the toxin-PDI complex and the samples were analyzed as in Fig. 1 B (lanes 5–7).

Figure 3.

Figure 3.

Release activity is present in Ero1α-containing fractions. (A) The protein in an ER extract was bound to a Q-Sepharose column and eluted with a continuous salt gradient. Fractions were analyzed for its Ero1 content by SDS-PAGE and immunoblotting with antibodies that either recognize both Ero1 isoforms (top), Ero1α only (middle), or Ero1β only (bottom). (B) The fractions were tested for their ability to induce toxin release, as described in Fig. 1 B.

Figure 4.

Figure 4.

Ero1 oxidizes the COOH-terminal disulfide bond in PDI and acts preferentially on the toxin–PDI complex. (A) Isolated A subunit (70 nM) and purified mammalian PDI (3 μM) were incubated in the presence of DTT (100 mM), GSSG (100 mM), or GSH (1 mM). Where indicated, purified mammalian Ero1α (0.3 μM or 3 μM) was added. 1/10 of the sample was TCA precipitated, washed with acetone, and treated with maleimide-PEG5000. The samples were subsequently analyzed by nonreducing SDS-PAGE followed by immunoblotting with an antibody against PDI. Arrows point to the number of cysteines in PDI modified with maleimide-PEG5000 (-SH-MP). (B) As in A except that purified yeast Ero1(0.3 μM or 1.5 μM) was used. (C) As in B except that 30 μM yeast Ero1 was added. (D) As in C except that 3 μM yeast mutant PDICxxC-AxxA or PDIAxxA-CxxC were used. (E) As in B except 70 nM or 3 μM of isolated A subunit were incubated with PDI (3 μM).

Figure 5.

Figure 5.

PDI-unfolded A1 peptide is transferred to the ER membrane. (A) Isolated A subunit (70 nM) was incubated in the presence of GSH (1 mM) with ER extract, proteoliposomes, or ER extract followed by proteoliposomes (lanes 1–6). After sedimentation, the proteoliposome-bound toxin in the pellet fraction and the unbound fraction in the supernatant were analyzed by nonreducing SDS-PAGE followed by immunoblotting. Controls were performed with either native microsomes (lanes 9 and 10), liposomes lacking proteins (lanes 7 and 8), or proteoliposomes generated from a proteinase K-treated detergent extract (lanes 15 and 16). (B) Isolated A subunit (70 nM) was incubated with either BSA, ER extract, or 6M urea (lanes 1–6). Where indicated, trypsin (100 μg/ml) was added. Samples were analyzed as in A. In lanes 7–10, isolated A subunit (70 nM) was incubated with either ER extract or 6M urea (followed by addition of proteoliposomes. Samples were analyzed as in A. (C) In lanes 1–3, ER extract or PDI-depleted extract were analyzed by immunoblotting with PDI antibodies. In lanes 4–7, isolated A subunit (70 nM) was incubated with these extracts, followed by the addition of proteoliposomes and the samples were separated into pellet and supernatant fractions. In lanes 8–11, isolated A subunit (70 nM) was incubated with purified PDI or BSA followed by addition of proteoliposomes. (D) As in Fig. 1 C, except BSA, wild-type mammalian proteoliposomes, wild-type yeast proteoliposomes, or yeast mutant ero1–1 proteoliposomes were added to the preformed PDI–toxin complex.

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