Unusual transfer of CutA into the secretory pathway, evidenced by fusion proteins with acetylcholinesterase.
The mouse CutA protein exists as long and short components of 20 and 15 kDa, produced by the use of different in-frame ATGs initiation codons, and by proteolytic cleavage. We recently showed that, surprisingly, the longer, uncleaved component resides mostly in the secretory pathway and is secreted, whereas the shorter component resides mostly in the cytoplasm. To confirm these subcellular localizations, we constructed fusion proteins in which the catalytic domain of rat acetylcholinesterase was placed downstream of the CutA variants. The acquisition of an active conformation and N-glycosylation of the fusion proteins proved their transfer into the secretory pathway. We show that the CutA-AChE fusion proteins produced and secreted active, N-glycosylated molecules, while an AChE mutant lacking its secretory signal peptide did not produce any significant activity. Thus, an N-terminal CutA domain actually drives AChE into the endoplasmic reticulum and allows its secretion. This was observed with full length CutA, starting at Met1, and at a much lower level with the shorter mutants starting at Met24 and Met44, although the latter is not predicted to possess any signal peptide. These experiments illustrate the value of using AChE as a reporter and reveals an unusual protein trafficking and secretory process.
Liang
Dong
D
Laboratoire de Neurobiologie, CNRS UMR 8544, Paris, France.
Carvalho
Stéphanie
S
Bon
Suzanne
S
Massoulié
Jean
J
D000110:Acetylcholinesterase / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D020134:Catalytic Domain; D002460:Cell Line; D006031:Glycosylation; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D051379:Mice; D021382:Protein Sorting Signals; D021381:Protein Transport; D011506:Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D055571:Secretory Pathway
C543918:CutA protein, mouse; D008565:Membrane Proteins; D021382:Protein Sorting Signals; D011506:Proteins; D011993:Recombinant Fusion Proteins; D000110:Acetylcholinesterase