
Analysis of random PCR‐originated mutants of the yeast Ste2 and Ste3 receptors
Author(s) -
Gastaldi Serena,
Zamboni Michela,
Bolasco Giulia,
Di Segni Gianfranco,
TocchiniValentini Glauco P.
Publication year - 2016
Publication title -
microbiologyopen
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.881
H-Index - 36
ISSN - 2045-8827
DOI - 10.1002/mbo3.361
Subject(s) - receptor , mutant , saccharomyces cerevisiae , yeast , gene , transmembrane domain , biology , extracellular , signal transduction , transmembrane protein , biochemistry , microbiology and biotechnology
The G protein‐coupled receptors Ste2 and Ste3 bind α‐ and a ‐factor, respectively, in Saccharomyces cerevisiae . These receptors share a similar conformation, with seven transmembrane segments, three intracellular loops, a C‐terminus tail, and three extracellular loops. However, the amino acid sequences of these two receptors bear no resemblance to each other. Coincidently the two ligands, α‐ and a ‐factor, have different sequences. Both receptors activate the same G protein. To identify amino acid residues that are important for signal transduction, the STE 2 and STE 3 genes were mutagenized by a random PCR ‐based method. Mutant receptors were analyzed in MAT α cells mutated in the ITC 1 gene, whose product represses transcription of a ‐specific genes in MAT α . Expression of STE 2 or STE 3 in these cells results in autocrine activation of the mating pathway, since this strain produces the Ste2 receptor in addition to its specific ligand, α‐factor. It also produces a ‐factor in addition to its specific receptor, Ste3. Therefore, this strain provides a convenient model to analyze mutants of both receptors in the same background. Many hyperactive mutations were found in STE 3 , whereas none was detected in STE 2 . This result is consistent with the different strategies that the two genes have adopted to be expressed.