N-terminal extension of the cholera toxin A1-chain causes rapid degradation after retrotranslocation from endoplasmic reticulum to cytosol

Abstract

Cholera toxin travels from the plasma membrane to the endoplasmic reticulum of host cells, where a portion of the toxin, the A1-chain, is unfolded and targeted to a protein-conducting channel for retrotranslocation to the cytosol. Unlike most retrotranslocation substrates, the A1-chain escapes degradation by the proteasome and refolds in the cytosol to induce disease. How this occurs remains poorly understood. Here, we show that an unstructured peptide appended to the N terminus of the A1-chain renders the toxin functionally inactive. Cleavage of the peptide extension prior to cell entry rescues toxin half-life and function. The loss of toxicity is explained by rapid degradation by the proteasome after retrotranslocation to the cytosol. Degradation of the mutant toxin does not follow the N-end rule but depends on the two Lys residues at positions 4 and 17 of the native A1-chain, consistent with polyubiquitination at these sites. Thus, retrotranslocation and refolding of the wild-type A1-chain must proceed in a way that protects these Lys residues from attack by E3 ligases.

FIGURE 1.

Functional analysis of HA-CT and CT-HA. A and B, schematics of the N- and C-terminal peptide extensions are shown. Arg-192 (R192) is located at the cleavage site separating the A1- and A2-chains and bridged by a disulfide bond. Lys-4 and Lys-17 are indicated. * indicates the cleavage site for AcTev protease. C, time course of CT-induced Cl⁻ secretion (I_sc) induced by WT CT (filled square), HA-CT (filled triangle), or HA-CT pretreated with AcTev protease to remove the HA tag (filled circle). Some monolayers were pretreated with brefeldin A (indicated by + BFA) followed by application of WT CT (open square) or CT-HA pretreated with AcTev protease to remove the HA tag (open triangle). The viability of monolayers was demonstrated by applying the cAMP agonist, forskolin. D, exactly as for panel C but using the mutant toxin CT-HA. Error bars in C and D indicate S.D.

FIGURE 2.

N-terminally tagged A1-chain does not appear to retrotranslocate from ER to cytosol. Retrotranslocation is measured in Vero cells by isolating cytosol from cell membranes and subcellular organelles via centrifugation. The presence of the A1-chain in the cytosolic fraction is analyzed by SDS-PAGE and immunoblot (IB) using antibodies recognizing the A-subunit (CTA) and B-subunit (CTB) or HA tag. A, retrotranslocation results are shown for WT CT (lane 4), the enzymatically inactive E112D mutant (lane 5), and the non-cleavable CT mutant R192G (lane 6). Lanes 1–3 are controls, and lanes 7–9 are the membrane fraction. B, SDS-PAGE and immunoblot of cytosolic and membrane fractions of the retrotranslocation assays using an antibody recognizing ER lumenal marker BiP. C, retrotranslocation assay using WT CT, HA-CT, and CT-HA as indicated. Lanes 1 and 12–14 are controls. * indicates an unidentified band. BFA, brefeldin A.

FIGURE 3.

HA-CT A1-chain is rapidly degraded by the proteasome. A, cells were pretreated with lactacystin or vehicle alone before performing retrotranslocation experiments. Cytosolic fractions were analyzed by SDS-PAGE and immunoblotted (IB) using antibodies recognizing the A-subunit (CTA) and B-subunit (CTB) or HA tag. Lanes 1 and 2 are controls. B, in a time course of CT-induced Cl⁻ secretion (I_sc), T84 cells were incubated with WT CT (filled square) or HA-CT (filled triangle) or pretreated with lactacystin followed by WT CT (open square) or HA-CT (open triangle). The viability of monolayers was demonstrated by applying the cAMP agonist, forskolin. C, summarized data from two independent experiments, each done in triplicate, of maximal I_sc for untreated or lactacystin (Lac) pretreated T84 cells is shown. Error bars in C and D indicate S.D.

FIGURE 4.

Mutation of lysines in the native A1-chain rescues HA-CT from the proteasome. A, cells were pretreated with lactacystin or vehicle alone before retrotranslocation experiments. WT toxin (lanes 6 and 7) and carbamylated WT CT with Lys-4 and Lys-17 mutations (WT K4RK17R*) (lanes 8 and 9) are shown. HA-CT (lanes 10 and 11), HA-CT K4RK17R (lanes 12 and 13), or the carbamylated HA-CT K4RK17R mutant (HA-CT K4RK17R*) (lanes 14 and 15) are shown. Lanes 1–5 are controls. All samples were analyzed by SDS-PAGE and immunoblotted (IB) using antibodies recognizing the A-subunit (CTA) and B-subunit (CTB) or HA tag. B, in a time course of CT-induced Cl⁻ secretion (I_sc), T84 cells were either treated with HA-CT K4RK17R* (filled circle) or pretreated with lactacystin followed by HA-CT K4RK17R* (open circle). C, this was performed as in panel B, but HA-CT K4RK17R (filled diamond) or pretreated HA-CT K4RK17R (open diamond) was tested. D, summarized data from three independent experiments, each done in triplicate, of rescue by lactacystin is shown. The percentage of increase in maximal I_sc for lactacystin-pretreated T84 cells versus untreated cells was plotted on the y axis for both HA-CT K4RK17R and HA-CT K4RK17R*. Error bars in indicate S.D.

FIGURE 5.

Development and functional analysis of HSV-CT. A, a schematic of the N-terminal peptide extensions, HA-CT and HSV-CT, is shown. Arg-192 (R192) is located at the cleavage site separating the A1- and A2-chains. A disulfide bond, in addition to other non-covalent interactions, links together the two chains. B, in a time course of CT-induced Cl⁻ secretion (I_sc), T84 cells were incubated with HSV-CT (filled circle) or HSV-CT pretreated with AcTev protease to remove the tag (HSV-CT, open circle). C, this was performed as in panel B, but cells were treated with HSV-CT (filled circle) or lactacystin-pretreated HSV-CT (open circle). Error bars in B and C indicate S.D.

FIGURE 6.

HA-CT refolds poorly after release from PDI in vitro. Briefly, we incubated all toxins with GM1-coupled beads and PDI in reducing conditions. PDI binds, unfolds, and releases the A1-chain into the supernatant from the B-subunit-GM1 magnetic beads. The supernatants were left untreated or were treated with GSSG to induce release of the A1-chain from PDI, and refolding was measured as resistance to trypsin degradation. All samples were analyzed by SDS-PAGE under non-reducing conditions, except the 4-ng control, which was reduced to separate the A1-chain. A, the unfolding and refolding of HA-CT (upper panel) and CT-HA (lower panel) were analyzed by immunoblot (IB) using an antibody against the HA tag. * indicates partially refolded toxin fragments. B, summarized data from four independent experiments. Compare HA-CT (white bars) with CT-HA (black bars). Band intensities were acquired using GeneSnap (GeneGnome HR, Syngene) and normalized to the total fraction of A1-chain released by PDI in the absence of trypsin (lanes 4). Error bars indicate S.D.