Wernick, Naomi B.Luca, Heidi DeKam, Wendy R.Lencer, Wayne I.2019-10-052010Wernick, Naomi L. B., Heidi De Luca, Wendy R. Kam, and Wayne I. Lencer. 2010. “N-Terminal Extension of the Cholera Toxin A1-Chain Causes Rapid Degradation after Retrotranslocation from Endoplasmic Reticulum to Cytosol.” Journal of Biological Chemistry 285 (9): 6145–52. https://doi.org/10.1074/jbc.m109.062067.0021-92581083-351Xhttp://nrs.harvard.edu/urn-3:HUL.InstRepos:41483332Cholera 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.en-USJournal Article2019-10-0510.1074/jbc.M109.062067