Yu, Lei and Blumer, Kendall J. and Davidson, Norman and Lester, Henry A. and Thorner, Jeremy (1989) Functional expression of the yeast alpha-factor receptor in Xenopus oocytes. Journal of Biological Chemistry, 264 (35). pp. 20847-20850. ISSN 0021-9258 http://resolver.caltech.edu/CaltechAUTHORS:YULjbc89
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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.
|Additional Information:||Copyright © 1989 by the American Society for Biochemistry and Molecular Biology. (Received for publication, June 19, 1989) We thank Susan Porter for the isolation of the yeast GPA1 gene. This work was supported by Grants NS11756, GM10991, and GM21841 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. [L.Y. was] [s]upported by a Del Webb postdoctoral fellowship. [K.J.M. was] [s]upported by American Cancer Society Postdoctoral Fellowship PF2632.|
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