Drosophila melanogaster acetylcholinesterase: identification and expression of two mutations responsible for cold- and heat-sensitive phenotypes.

AceIJ29 and AceIJ40 are cold- and heat-sensitive variants of the gene coding for acetylcholinesterase in Drosophila melanogaster. In the homozygous condition, these mutations are lethal when animals are raised at restrictive temperatures, i.e., below 23 degrees C for AceIJ29 or above 25 degrees C for AceIJ40. The coding regions of the gene in these mutants were sequenced and mutations changing Ser374 to Phe in AceIJ29 and Pro75 to Leu in AceIJ40 were found. Acetylcholinesterases bearing these mutations were expressed in Xenopus oocytes and we found that these mutations decrease the secretion rate of the protein most probably by affecting its folding. This phenomenon is exacerbated at restrictive temperatures decreasing the amount of secreted acetylcholinesterase below the lethality threshold. In parallel, the substitution of the conserved Asp248 by an Asn residue completely inhibits the activity of the enzyme and its secretion, preventing the correct folding of the protein in a non-conditional manner.