The Leu‐132 of the Ste4(Gβ) subunit is essential for proper coupling of the G protein with the Ste2 α factor receptor during the mating pheromone response in yeast

Abstract

In order to identify amino acid residues of Ste4p involved in receptor recognition and/or receptor-G protein coupling, we employed random in vitro mutagenesis and a genetic screening to isolate mutant Ste4p subunits with altered pheromone response. We generated a plasmid library containing randomly mutagenized Ste4 ORFs, followed by phenotypic selection of ste4p mutants by altered K K pheromone response in yeast cells. Subsequently, we analyzed mutant ste4-10 which has a replacement of the almost universally conserved leucine 132 by phenylalanine. This residue lies in the first blade of the L L propeller structure proposed by crystallographic analysis. By overexpression experiments we found that mutant ste4p subunit triggers the mating pathway at wild type levels in both wild type and receptorless strains. When expressed in a ste4 background, however, the mutant G protein is activated inefficiently by mating pheromone in both a and K K cells. The mutant ste4-10p was tested in the two-hybrid system and found to be defective in its interaction with the Gpa1p, but has a normal association with the C-termini end of the Ste2p receptor. These observations strongly suggest that the Leu-132 of the Ste4p subunit is essential for efficient activation of the G protein by the pheromonestimulated receptor and that this domain could be an important point for physical interaction between the GL L and the GK K subunits.z 2000 Federation of European Biochemical Societies.

The KlGpa1 Gene Encodes a G-Protein α Subunit That Is a Positive Control Element in the Mating Pathway of the Budding Yeast Kluyveromyces lactis

“…While no significant effect was observed in a qualitative experiment of diploid formation, i.e., S. cerevisiae cells carrying either KlGpa1 or KlGpa2 mate normally, we observed a weak effect of KlGpa1p on the expression of Fus1 measured with the Fus1-LacZ fusion (21). Wild-type cells expressing KlGpa1 in a multicopy plasmid show 1.5-times-lower ␤-galactosidase activity than cells carrying the vector alone.…”

“…Assays of physical interaction were done with the LexA-B42 two-hybrid systems as described previously (7). S. cerevisiae Gpa1 and Ste4 genes were subcloned into pJG4-5 and pEG202 as described previously (21). KlGpa1 and KlGpa2 were amplified by PCR, introducing NcoI (position Ϫ1) and EcoRI (position Ϫ5)/BamHI (18 nucleotides after the stop codon) sites, respectively.…”

“…Expression of Fus1-LacZ fusion was done as described previously (21). Molecular biology procedures were performed as described by Sambrook et al (24).…”

The KlSTE2 and KlSTE3 genes encode MATα‐ and MATa‐specific G‐protein‐coupled receptors, respectively, which are required for mating of Kluyveromyces lactis haploid cells

“…Furthermore, this property was used as practical interaction screening for cloning the Gpa2-coupled receptor in S. cerevisiae (CheolWon et al, 1997). Interaction of the C-terminus of KlSte3p with the various G protein subunits was achieved by the two-hybrid system (Ongay-Larios et al, 2000). In this experiment we measured the β-galactosidase activity induced by the magnitude of the interaction between proteins (Figure 4).…”

“…Assays of physical interaction were performed with the LexA-B42 two-hybrid system as described (Ongay-Larios et al, 2000). The fragment encoding the C-terminus tail of KlSte3p was digested with the restriction enzyme SspI and cloned inframe into pJG4-5.…”