The C-terminal T peptide of acetylcholinesterase enhances degradation of unassembled active subunits through the ERAD pathway.
The catalytic domain of acetylcholinesterase AChE(T) subunits is followed by a C-terminal T peptide which mediates their association with the proline-rich attachment domain (PRAD) of anchoring proteins. Addition of the T peptide induced intracellular degradation and concomitantly reduced to variable degrees the secretion of AChE species differing in their oligomerization capacity and of human alkaline phosphatase. The T peptide forms an amphiphilic alpha-helix, containing a series of conserved aromatic residues. Replacement of two, four or five aromatic residues gradually suppressed degradation and increased secretion. Co-expression with a PRAD- containing protein induced the assembly of PRAD-linked tetramers in the endoplasmic reticulum (ER) and allowed partial secretion of a dimerization- defective mutant; by masking the aromatic side chains, hetero-oligomerization rescued this enzyme from degradation. Degradation was due to ERAD, since it was not blocked by brefeldin A but was sensitive to proteasome inhibitors. Kifunensine reduced degradation, suggesting a cooperativity between the glycosylated catalytic domain and the non-glycosylated T peptide. This system appears particularly well suited to analyze the mechanisms which determine the degradation of correctly folded multidomain proteins in the ER.
Belbeoc'h
Stéphanie
S
Laboratoire de Neurobiologie Cellulaire et Moléculaire, CNRS UMR 8544, Ecole Normale Supérieure, 46 rue d'Ulm, 75230 Paris Cedex 05, France.
Massoulié
Jean
J
Bon
Suzanne
S
D000110:Acetylcholinesterase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000469:Alkaline Phosphatase / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D024322:Amino Acids, Aromatic / Q000737:chemistry / Q000235:genetics; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D017124:Conserved Sequence; D003545:Cysteine / Q000378:metabolism; D019281:Dimerization; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006801:Humans; D007783:Lactones / Q000494:pharmacology; D007976:Leupeptins / Q000494:pharmacology; D008969:Molecular Sequence Data; D009559:Nippostrongylus / Q000201:enzymology; D010455:Peptides / Q000378:metabolism*; D021122:Protein Subunits / Q000378:metabolism*; D051381:Rats; D012694:Serine / Q000378:metabolism
D024322:Amino Acids, Aromatic; D007783:Lactones; D007976:Leupeptins; D010455:Peptides; D021122:Protein Subunits; C098869:clasto-lactacystin beta-lactone; D012694:Serine; D000110:Acetylcholinesterase; D000469:Alkaline Phosphatase; D003545:Cysteine; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde