Expression of Torpedo nicotinic acetylcholine receptor subunits in yeast is enhanced by use of yeast signal sequences.
We have produced the four subunits of the nicotinic acetylcholine receptor of Torpedo californica, an integral membrane protein, in the yeast Saccharomyces cerevisiae. Two of the subunits (alpha and delta) were readily produced from their cDNAs after simply subcloning them into a yeast shuttle vector adjacent to a yeast promoter. The other two protein subunits (beta and gamma) were not produced by this strategy, although the amounts of mRNA produced from these expression constructs are similar to those for alpha and delta. Replacing the DNA coding for the normal N-terminal signal sequences for the beta and gamma subunits with DNA coding for the signal sequence of yeast invertase results in successful protein synthesis. The yeast signal sequence allows these subunits to be translocated across the membrane of the endoplasmic reticulum and to be glycosylated. The appropriate final size of the subunit proteins suggests that the yeast signal sequence has been properly cleaved after translocation.
Yellen
G
G
Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
Migeon
J C
JC
D000818:Animals; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000235:genetics; D015870:Gene Expression; D006031:Glycosylation; D046911:Macromolecular Substances; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics*; D011978:Receptors, Nicotinic / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013728:Terminator Regions, Genetic; D014101:Torpedo
D046911:Macromolecular Substances; D021382:Protein Sorting Signals; D011978:Receptors, Nicotinic; D004247:DNA