N-terminal extension of the cholera toxin A1-chain causes rapid degradation after retrotranslocation from endoplasmic reticulum to cytosol.
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.
Wernick
Naomi L B
NL
Gastrointestinal Cell Biology Laboratory, Children's Hospital, and Harvard Medical School, Boston, Massachusetts 02115, USA.
De Luca
Heidi
H
Kam
Wendy R
WR
Lencer
Wayne I
WI
D000818:Animals; D002522:Chlorocebus aethiops; D002772:Cholera Toxin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006207:Half-Life; D008239:Lysine; D010455:Peptides / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D055550:Protein Stability; D021381:Protein Transport; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D014709:Vero Cells
D010455:Peptides; D002772:Cholera Toxin; D044767:Ubiquitin-Protein Ligases; D046988:Proteasome Endopeptidase Complex; D008239:Lysine