Functional expression of the yeast alpha-factor receptor in Xenopus oocytes.
The STE2 gene of the yeast Saccharomyces cerevisiae encodes a 431-residue polypeptide that has been shown by chemical cross-linking and genetic studies to be a component of the receptor for the peptide mating pheromone, alpha-factor. To demonstrate directly that the ligand binding site of the alpha-factor receptor is comprised solely of the STE2 gene product, the STE2 protein was expressed in Xenopus oocytes. Oocytes microinjected with synthetic STE2 mRNA displayed specific surface binding for 35S-labeled alpha-factor (up to 40 sites/micron2/ng RNA). Oocytes injected with either STE2 antisense RNA or heterologous receptor mRNA (nicotinic acetylcholine receptor alpha, beta, gamma, and delta subunit mRNAs) showed no binding activity (indistinguishable from uninjected control oocytes). The apparent KD (7 nM) of the alpha-factor binding sites expressed on the oocyte surface, determined by competition binding studies, agreed with the values reported for intact yeast cells and yeast plasma membrane fractions. These findings demonstrate that the STE2 gene product is the only yeast polypeptide required for biogenesis of a functional alpha-factor receptor. Electrophysiological measurements indicated that the membrane conductance of oocytes injected with STE2 mRNA, or with both STE2 and GPA1 (encoding a yeast G protein alpha-subunit) mRNAs, did not change and was not affected by pheromone binding. Thus, the alpha-factor receptor, like mammalian G protein-coupled receptors, apparently lacks activity as an intrinsic or ligand-gated ion channel. This report is the first instance in which a membrane-bound receptor from a unicellular eukaryote has been expressed in a vertebrate cell.
Yu
L
L
Division of Biology, California Institute of Technology, Pasadena 91125.
Blumer
K J
KJ
Davidson
N
N
Lester
H A
HA
Thorner
J
J
D000818:Animals; D005260:Female; D015870:Gene Expression; D005800:Genes, Fungal; D007700:Kinetics; D000072235:Mating Factor; D009865:Oocytes / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D010675:Pheromones / Q000378:metabolism; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics; D011956:Receptors, Cell Surface / Q000096:biosynthesis / Q000235:genetics*; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013698:Templates, Genetic; D014157:Transcription Factors; D014158:Transcription, Genetic; D014981:Xenopus
D010455:Peptides; D010675:Pheromones; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide; D014157:Transcription Factors; D000072235:Mating Factor