N-terminal Extension of the Cholera Toxin A1-chain Causes Rapid Degradation after Retrotranslocation from Endoplasmic Reticulum to Cytosol |
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Authors: | Naomi L B Wernick Heidi De Luca Wendy R Kam and Wayne I Lencer |
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Institution: | From the ‡Gastrointestinal Cell Biology Laboratory, Children''s Hospital, and Harvard Medical School and ;the §Harvard Digestive Diseases Center, Boston, Massachusetts 02115 |
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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. |
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Keywords: | Proteases/Ubiquitination Protein/Degradation Protein/Translocation Subcellular Organelles/Endoplasmic Reticulum Toxins Cholera Toxin Proteasome |
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