Analysis of random PCR-originated mutants of the yeast Ste2 and Ste3 receptors

Abstract

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 STE2 and STE3 genes were mutagenized by a random PCR-based method. Mutant receptors were analyzed in MATα cells mutated in the ITC1 gene, whose product represses transcription of a-specific genes in MATα. Expression of STE2 or STE3 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 STE3, whereas none was detected in STE2. This result is consistent with the different strategies that the two genes have adopted to be expressed.

Keywords: G protein-coupled receptors; hyperactive mutations; mating response; signal transduction.

Figure 1

Identification of Ste2 and Ste3 receptor mutants with reduced mating activity. (A) The autocrine M18 strain, harboring the STE2 or STE3 genes mutagenized by a random PCR‐based method, was plated on an X‐Gal medium (the plate shown is for STE2; similar plates were obtained for STE3). Colonies appearing white or light‐blue, as opposed to the deep blue of colonies containing nonmutagenized genes, were picked up and patched again to confirm the colony phenotype. (B) and (C) Quantitative β‐Gal assay of the indicated white or light‐blue mutants of, respectively, the Ste2 and Ste3 receptors. The results for each mutant represent the averages of three samples, normalized to the level of β‐Gal activity of cells expressing the nonmutagenized receptors; error bars correspond to 1 standard deviation (SD).

Figure 2

Distribution of the mutations in the Ste2 or Ste3 receptors. The predicted topological structures of the seven transmembrane segments of the Ste2 and Ste3 receptors are shown. Only the amino acids subjected to random mutagenesis are depicted as circles. The relative β‐Gal activity induced by the mutated full‐length receptors was determined in strain RM6 (for Ste2) or DDS4 (for Ste3). Mutated residues are colored as indicated.

Figure 3

Mutants of the Ste2 receptor with lower activity in a nonautocrine strain. The position of mutated residues is depicted on the top. Plasmids coding for the indicated mutant or wild‐type Ste2 receptors were introduced into two different strains, the nonautocrine RM6 (MAT a) and the autocrine DDS4 (MATα strain). RM6 cells were incubated for 4 h together with a cell‐free broth of an overnight culture of MATα strain GDS30, as a source of α‐factor. Autocrine DDS4 cells were incubated for 4 h in the absence of exogenous α‐factor. The results for each mutant represent the average of three samples, normalized to the level of β‐Gal activity of cells expressing the nonmutagenized receptors; error bars correspond to 1 SD. The horizontal line shows the value for wild‐type receptor.

Figure 4

Analysis of selected down mutations of the Ste2 receptor in different strains. (A) The position of mutated residues of the Ste2 receptor is depicted on the top. The β‐Gal activity of different strains carrying plasmids coding for the indicated mutants is shown. Data are normalized with respect to wild type. The main features of the strains are indicated. The sst2 mutation confers supersensitivity; the itc1 mutation permits the expression of the BAR1 protease, therefore decreasing the amount of α‐factor. (B) Western blot. The RM6 strain was transformed with plasmids coding for the indicated null mutant or wild‐type (WT) receptors, or with the empty vector (V). Total cell lysates (30 μg each sample) were loaded on polyacrylamide gels and analyzed by western blot using anti‐HA antibodies. Protein standards are shown in lane M. (C) Confocal microscopy analysis on Ste2‐GFP (WT) and mutant Ste2‐S207P‐GFP in RM6 strain, two samples each. Wild‐type Ste2‐GFP receptor (green) appears to be localized at the cell membrane and in vesicles around the vacuolar lumen (magenta). In S207P mutant, GFP signal (green) seems localized at the perivacuolar area (magenta), whereas it appears less intense at the cell membrane. Not in all cells the vacuole was visible. GFP, green fluorescent protein.

Figure 5

Analysis of selected mutations in the extracellular loops of the Ste3 receptor in different strains. (A) The position of mutated residues of the Ste3 receptor is depicted on the top. The β‐Gal activity of different strains carrying plasmids coding for the indicated mutants is shown. Data are normalized with respect to wild type. The horizontal line shows the value for the wild‐type receptor. The main features of the strains are indicated. The itc1 mutation confers an autocrine character for the Ste3 receptor. (B) Western blot. The DDS4 strain was transformed with plasmids coding for the indicated mutant or wild‐type (WT) receptors, or with the empty vector (V). Total cell lysates (20 μg each sample) were loaded on polyacrylamide gels and analyzed as indicated in the legend of Fig. 4.

Figure 6

Confocal microscopy analysis on Ste3‐GFP (WT) and mutant Ste3‐C147Y‐GFP in DDS4, DDS2, and M18 strain, two samples each. Wild‐type Ste3‐GFP receptor (green) appears to be localized at the cell membrane and in vesicles around the vacuolar lumen (magenta). In C147Y mutant, GFP signal (green) is localized in vesicles throughout the cytoplasm and at the perivacuolar area (magenta). Not in all cells the vacuole was visible. GFP, green fluorescent protein.

Figure 7

Hyperactive mutants of the Ste3 receptor. (A) The position of mutated residues with relative β‐Gal activity higher than the wild‐type Ste3 receptor is depicted on the top. The activity of different strains carrying plasmids coding for the indicated mutants is shown. Data are normalized with respect to wild type. The horizontal line shows the value for the wild‐type receptor. The main features of the strains are indicated. Autocrine M18 cells were incubated for 2.5 h in the absence of exogenous a‐factor: short and full‐length mutant receptors were analyzed, as indicated. DDS4 and DDS2 cells were incubated for 4 h together with a cell‐free broth of an overnight culture of MAT a strain GDS31, as a source of a‐factor. Hyperactivity is detected only in DDS4 cells. (B) Confocal microscopy analysis on Ste3‐GFP (WT) and hyperactive mutant Ste3‐T197I‐GFP in DDS4 strain. Mutant receptor (green) is highly expressed and localized at the cell membrane and throughout the cytoplasm. (C) Western blot. Total cell lysates (20 μg each sample) of DDS4 cells transformed with plasmids coding for the indicated mutant or wild‐type (WT) receptors, or with the empty vector (V), were loaded on polyacrylamide gels and analyzed as indicated in the legend of Fig. 4. GFP, green fluorescent protein.