N-terminal Extension of the Cholera Toxin A1-chain Causes Rapid Degradation after Retrotranslocation from Endoplasmic Reticulum to Cytosol

Naomi L.B Wernick,Heidi De Luca,Wendy R Kam,Wayne I Lencer

doi:10.1074/jbc.m109.062067

Naomi L.B Wernick, Heidi De Luca + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m109.062067

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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.

Highlights

Structure and Function of the Toxin Mutants—Two mutant toxins were prepared containing the HA epitope linked to the N or C terminus of the A1-chain by a tobacco etch virus protease cleavage site (TEV) and flanked at both ends by Gly and Ser residues (Fig. 1, A and B, named HA-cholera toxin (CT) and CT-HA, respectively, and see supplemental Fig. 1)
Toxin function was examined using intestinal T84 cells grown in monolayer culture, which respond to CT by producing a cAMP-dependent ClϪ secretory response
Most endoplasmic reticulum (ER)-associated degradation (ERAD) substrates in mammalian cells and yeast are retrotranslocated to the cytosol and targeted to the proteasome by polyubiquitination [22]

Summary

Introduction

The assay is robust response, indicating that both mutant toxins required retro- and specific for measuring retrotranslocation of the grade vesicular transport from plasma membrane to ER to A1-chain (Fig. 2A, compare middle and right panels). HA-CT mutant (Fig. 2C, middle panel, compare lanes 4 and 5), Retrotranslocation of the A1-chain in Intact Cells—To under- consistent with rescue of toxin function by TEV protease (Fig. stand why the N-terminal mutant lacked activity, we modified 1C).

Results

Conclusion