The Ero1alpha-PDI redox cycle regulates retro-translocation of cholera toxin

Abstract

Cholera toxin (CT) is transported from the plasma membrane of host cells to the endoplasmic reticulum (ER) where the catalytic CTA1 subunit retro-translocates to the cytosol to induce toxicity. Our previous analyses demonstrated that the ER oxidoreductase protein disulfide isomerase (PDI) acts as a redox-dependent chaperone to unfold CTA1, a reaction postulated to initiate toxin retro-translocation. In its reduced state, PDI binds and unfolds CTA1; subsequent oxidation of PDI by Ero1alpha enables toxin release. Whether this in vitro model describes events in cells that control CTA1 retro-translocation is unknown. Here we show that down-regulation of Ero1alpha decreases retro-translocation of CTA1 by increasing reduced PDI and blocking efficient toxin release. Overexpression of Ero1alpha also attenuates CTA1 retro-translocation, an effect due to increased PDI oxidation, which prevents PDI from engaging the toxin effectively. Interestingly, Ero1alpha down-regulation increases interaction between PDI and Derlin-1, an ER membrane protein that is a component of the retro-translocation complex. These findings demonstrate that an appropriate Ero1alpha-PDI ratio is critical for regulating the binding-release cycle of CTA1 by PDI during retro-translocation, and implicate PDI's redox state in targeting it to the retro-translocon.

Figure 1.

Ero1α down-regulation decreases retro-translocation of CTA1. (A) 293T cells were transfected with a scrambled siRNA (lane 1) or an Ero1α-specific siRNA (lane 2). Cells were harvested and lysed, and the lysates were subjected to immunoblot analysis with the indicated antibodies. Lanes 3–6, RT-PCR analysis of the unspliced (u) and spliced (s) forms of the XBP1 mRNA from cells treated with DTT or tunicamycin or from cells transfected with a scrambled or Ero1α-specific siRNA. (B) Cells transfected with a scrambled or an Ero1α-specific siRNA were treated with 10 nM CT and subjected to the retro-translocation assay. Supernatant and pellet fractions were analyzed by nonreducing SDS-PAGE, followed by immunoblotting with the indicated antibodies. CTA is 28 kDa and CTA1 is 22 kDa. (C) The intensity of the CTA1 band generated in B was quantified with ImageJ (NIH; http://rsb.info.nih.gov/ij/). Mean ± SD of at least three independent experiments is shown. A two-tailed t test was used. (D) Cells transfected with a scrambled or Ero1α-specific siRNA were treated with 10 nM CT, and the cAMP level was measured with a cAMP Biotrak Enzyme Immunoassay System (GE Healthcare). Data were normalized against the forskolin-induced cAMP level, as demonstrated previously (Forster et al., 2006). Mean ± SD of at least three independent experiments is shown. A two-tailed t test was used. (E) 293T cells transiently expressing TCRα were transfected with a scrambled or an Ero1α-specific siRNA, labeled with [³⁵S]methionine, and harvested at the indicated chase times, and the resulting cell lysate was used for TCRα immunoprecipitation. Signals were detected by autoradiography. Bottom panel, quantification of the intensity of the TCRα band from three independent experiments; values are expressed as a total percentage of the TCRα band at chase time = 0. Error bars, ±SD.

Figure 2.

Ero1α overexpression attenuates retro-translocation of CTA1. (A) Lanes 1–4, lysates from 293T cells transfected with vector, WT Ero1α, or Ero1α(C94A:C99A) were analyzed for expression of Ero1α, BiP, PDI, Derlin-1, and Hsp90. Lanes 5–8, RT-PCR analysis of the unspliced (u) and spliced (s) forms of the XBP1 mRNA from cells treated with DTT or tunicamycin or from cells transfected with vector or a WT Ero1α construct. (B) Cells in A were subjected to the retro-translocation assay as described in Figure 1. (C) Lysates from 293T cells transfected with vector, WT Ero1α, or WT Ero1α and PDI were analyzed for expression of Ero1α, PDI, and Hsp90. (D) Cells in C were subjected to the retro-translocation assay as in Figure 1. (E) Quantification of the CTA1 band intensity in B and D. Mean ± SD of at least three independent experiments is shown. A two-tailed t test was used. Results from overexpression of Ero1β and Derlin-1-YFP on CTA1 retro-translocation are also included.

Figure 3.

Altering Ero1α level in cells affects the redox state of PDI. (A) 293T cells were treated with NEM and lysed, and the endogenous PDI was immunoprecipitated from the lysate. The immunoprecipitate was incubated with TCEP, washed, and treated with or without MPEG. Samples were analyzed by SDS-PAGE and immunoblotted with an antibody against PDI. (B) 293T cells transfected with a scrambled or an Ero1α-specific siRNA were analyzed as in A, with the exception that both samples were treated with MPEG. (C) As in B, except ERp57 was immunoprecipitated and immunoblotted instead of PDI. (D) 293T cells transfected with vector or WT Ero1α were analyzed as in B.

Figure 4.

The level of Ero1α controls PDI–CTA1 interaction. (A) 293T cells were transfected with WT PDI FLAG and either a scrambled or an Ero1α-specific siRNA, followed by incubation of the cells with or without CT. DSP cross-linker was added to the cells, followed by lysis with 1% Triton X-100. A CTA-specific antibody was incubated with the resulting lysate, and the precipitated sample was subjected to reducing SDS-PAGE followed by immunoblotting with the indicated antibodies. (B) As in A, except no DSP was added, and DBC was used instead of Triton X-100. Samples were subjected to nonreducing SDS-PAGE. (C) Cells overexpressing WT PDI FLAG were treated with or without NEM 60 min after CT intoxication, and the PDI–CTA1 interaction was analyzed as in A. (D) As in A, except cells were transfected with either vector or WT Ero1α.

Figure 5.

Down-regulation of Ero1α increases PDI–Derlin-1 interaction. (A) 293T cells were transfected with WT PDI FLAG and either a scrambled or an Ero1α-specific siRNA. The cells were lysed with 1% DBC, and the lysate was incubated with either a control Myc or a Derlin-1–specific antibody. The immunoprecipitated sample was subjected to reducing SDS-PAGE and immunoblotted with the indicated antibodies. (B) As in A, except I272W PDI FLAG was transfected instead of WT PDI FLAG. (C) As in A, except Hrd1-Myc was transfected where indicated. (D) As in A, except no WT PDI FLAG was transfected, and endogenous p97 was immunoblotted. (E) Cells overexpressing WT PDI FLAG were treated with or without NEM 30 min before harvesting, and the PDI–Derlin-1 interaction analyzed as in A. (F) Cells were transfected with WT PDI FLAG and either vector or WT Ero1α, and the PDI–Derlin-1 interaction analyzed as in A. (G) Model of the Ero1α-driven redox-dependent binding of PDI to CTA1 and Derlin-1 (see text for discussion).