Derlin-1 facilitates the retro-translocation of cholera toxin

Abstract

Cholera toxin (CT) intoxicates cells by using its receptor-binding B subunit (CTB) to traffic from the plasma membrane to the endoplasmic reticulum (ER). In this compartment, the catalytic A1 subunit (CTA1) is unfolded by protein disulfide isomerase (PDI) and retro-translocated to the cytosol where it triggers a signaling cascade, leading to secretory diarrhea. How CT is targeted to the site of retro-translocation in the ER membrane to initiate translocation is unclear. Using a semipermeabilized-cell retro-translocation assay, we demonstrate that a dominant-negative Derlin-1-YFP fusion protein attenuates the ER-to-cytosol transport of CTA1. Derlin-1 interacts with CTB and the ER chaperone PDI as assessed by coimmunoprecipitation experiments. An in vitro membrane-binding assay showed that CTB stimulated the unfolded CTA1 chain to bind to the ER membrane. Moreover, intoxication of intact cells with CTB stabilized the degradation of a Derlin-1-dependent substrate, suggesting that CT uses the Derlin-1 pathway. These findings indicate that Derlin-1 facilitates the retro-translocation of CT. CTB may play a role in this process by targeting the holotoxin to Derlin-1, enabling the Derlin-1-bound PDI to unfold the A1 subunit and prepare it for transport.

Figure 1.

Derlin-1 facilitates the retro-translocation of CTA1. (A) 293T cells were incubated with CT (10 nM) for 90 min at 37°C with or without BFA. Cells were permeabilized and 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 kDa, respectively. (B) 293T cells expressing YFP, Derlin-1-YFP, or Derlin-2-YFP were harvested and lysed, and the lysates were subjected to SDS-PAGE and immunoblotted with a GFP antibody. (C) 293T cells expressing either YFP, Derlin-1-YFP, or Derlin-2-YFP were incubated with CT (10 nM) for 45 or 90 min, the cells were permeabilized and centrifuged, and the supernatant fraction was analyzed as in A. The intensity of the CTA1 band generated from cells treated with CT for 90 min was quantified with ImageJ. Mean ± SD (error bars) of two to five independent experiments is shown. (D) 293T cells expressing YFP or Derlin-1-YFP were incubated with CT for 90 min and the cAMP level was measured by a cAMP Biotrak Enzyme Immunoassay System (GE Healthcare). Data were normalized against the forskolin-induced cAMP level, as demonstrated previously (Forster et al., 2006).

Figure 2.

CTB interacts with Derlin-1 and enhances the transfer of CTA1 to the ER membrane. (A) 293T cells were incubated with or without CT (30 nM) for 90 min, and the cells were harvested and lysed in a buffer containing 1% Triton X-100. The lysates were subjected to immunoprecipitation using the indicated antibodies. The precipitated samples were analyzed by SDS-PAGE and immunoblotted with the indicated antibodies. (B) As in A, except cells were incubated with CT (10 nM), and where indicated, the Sec61 antibody was used instead of the ERp29 antibody. (C) As in A, except cells were incubated with either CT (10 nM) or CTB (10 nM). (D) CT (200 nM) and PDI (2 μg/ml) were incubated with or without proteoliposomes or with PK-RM, and the samples were solubilized and precipitated using the indicated antibodies. The samples were analyzed as in A. (E) As in D, except the samples were precipitated using Sec61 or Derlin-1 antibodies. (F) CTA or CT was incubated with proteoliposomes, PDI, and GSH. Samples were sedimented, and the supernatant and pellet fractions were analyzed in SDS-PAGE followed by immunoblotting with an antibody against CTA.

Figure 3.

Derlin-1-YFP binds to CT and imparts a structural change on Derlin-1. (A) 293T cells expressing YFP, Derlin-1-YFP, or Derlin-2-YFP were intoxicated with CT (10 nM), the cells were lysed in a buffer containing 1% Triton X-100, and the lysate was subjected to immunoprecipitation as in Figure 2A. (B) As in A, except cells were intoxicated with CTB (10 nM). (C) Digitonin-treated 293T cells or cells overexpressing Derlin-1-YFP or Derlin-1-HA were incubated with 0.1 or 1 mg/ml Proteinase K, subjected to SDS-PAGE, and immunoblotted with an antibody against Derlin-1, GFP, or HA. (D) As in C, except samples were immunoblotted with a Derlin-2 antibody.

Figure 4.

CTB stabilizes the Derlin-1–dependent retro-translocation substrate CFTR. (A) 293T cells transiently expressing CFTR were incubated with CTB (100 nM), labeled with [³⁵S]methionine, and harvested at the indicated chase times, and the resulting cell lysate was used for CFTR immunoprecipitation. Signals were detected by autoradiography. Bottom panel, quantification of the intensity of Bands B and C from three independent experiments; values expressed as a percentage of the total intensity of Band B and Band C at chase time = 0. Bars, SD. Band B, the immature core-glycosylated form of CFTR; Band C, the mature complex-glycosylated form. (B) RNA isolated from cells incubated with or without CTB (100 nM) was subjected to semiquantitative RT-PCR analysis. Shown is the result of 30 cycles of amplifications. A similar result was obtained with 27 cycles of amplifications. (C) The same lysates in B were analyzed for the presence of PDI, ERp72, and Derlin-1. (D) HeLa cells stably expressing NHK were incubated with CTB (100 nM), and the lysate was subjected to SDS-PAGE and immunoblotted with an antibody against NHK.

Figure 5.

Association of PDI with Derlins. (A) 293T cells were lysed in a buffer containing 1% deoxyBigChap, and the lysates were subjected to immunoprecipitation as in Figure 2A. (B) As in A, except HeLa cells were used. (C) As in A, except 293T cells expressing Derlin-1-YFP were used. (D) As in A, except 293T cells expressing either wild-type mouse PDI-FLAG or the substrate-binding I272W mutant PDI-FLAG were lysed in a buffer containing 1% Tween 20. (E) Model of the initiation of retro-translocation of cholera toxin. See text for discussion.