Signal transduction pathways with common components mediate two life cycle transitions in haploid S. cerevisiae. See text for explanation.

Location of phosphorylation sites in Ste7 and SDS-PAGE mobility of variant proteins with phosphorylation site substitution mutations. (A) Residues phosphorylated in the activation loop of the catalytic domain (C) and feedback sites phosphorylated by Fus3/Kss1 in the regulatory domains (R). (B and C) Western blots show mobility of Ste7 species that are characteristic of hyperphosphorylated (HP-Ste7) or hypophosphorylated (Ste7) forms after SDS-PAGE fractionation of protein extracts from uninduced (−) or induced (+) cultures as indicated. Residues at the activation loop (Loop) and feedback (FB) sites denote the different Ste7 variants. Ste7M variants were expressed from the CYC1 promoter on plasmids in strain C699-89 (ste7Δ) or C699-106 (ste7Δ) using the following plasmids: pNC318 (Ste7M), pNC586 (Ste7M-A₇), pNC892 (Ste7M-E₇), pNC589 (Ste7M-EE), pNC597 (Ste7M-EE-A₇), pNC893 (Ste7M-EE-E₇), pNC571 (Ste7M-AA), pNC894 (Ste7M-AA-A₇), and pNC895 (Ste7M-AA-E₇).

In vitro kinase activities of Ste7 phosphorylation site substitution derivatives. Upper panels show the PhosphorImage of ³²P incorporation into Fus3R⁴², and lower panels show immune blots detecting immune-precipitated Ste7 after SDS-PAGE fractionation of proteins from immune complex kinase assay reactions. Cultures for these analyses were either untreated (−) or treated with pheromone (+), as indicated. Activities for each sample are calculated as ([³²P]Fus3 signal)/(IP-Ste7 signal). The numbers below the immune blot report the activity for each reaction as a percentage of that obtained for the sample with pheromone-induced Ste7 (WT). (A) Samples in lanes 1 to 6 are from strain K2149 (FUS3KSS1), and those in lanes 7 to 9 are from strain K2313 (fus3Δ kss1Δ) expressing catalytically inactive Ste7M-R²²⁰ (R²²⁰; pNC318-R220), wild-type Ste7M (Wt; pNC318), or Ste7M that cannot be feedback phosphorylated (A₇; pNC586). (B) Samples in lanes 1 to 3, 6, and 7 are from strain K2149 (FUS3 KSS1), and those in lanes 4, 5, 8, and 9 are from strain K2313 (fus3Δ kss1Δ) expressing Ste7M (Wt; pNC318), constitutively active Ste7M (EE; pNC589), or constitutively active Ste7M that cannot be feedback phosphorylated (EE-A₇; pNC597).

Ubiquitin modification of Ste7 phosphorylation site variants. Western blots show mobility of Ste7 species that are characteristic of the ubiquitinated (Ubi-Ste7), hyperphosphorylated (HP-Ste7), and hypophosphorylated (Ste7) forms. (A) Ste7 proteins were expressed from the STE7 promoter in strain YHD1001 (ste7Δ ubp3Δ) by using plasmids pNC766 (Ste7M), pNC768 (Ste7M-EE), pNC770 (Ste7M-EE-A₇), pNC791 Ste7M-EE-E₇), pNC781 (Ste7M-AA-A₇), or pNC790 (Ste7M-AA-E₇). The same strain with vector (pRS316) was used as a negative reference. (B) Ste7 proteins were expressed from the CYC1 promoter in strain C699-32 (ste7Δ) or strain C699-81 (ste7Δ ste11Δ) carrying pYES-UBP3^C469S by using plasmids pNC318 (Ste7M) or pNC597 (Ste7M-EE-A₇). The same strain with vector (pNC160) was used as a negative reference.

Mating and pheromone response assays comparing the in vivo function of Ste7 phosphorylation site substitution derivatives. (A) Semiquantitative mating assays. Photographs show diploid colonies selected for growth on minimal medium after an overnight incubation of mating mixtures at 30°C. Tester strain KZ8-1D (MATα; 10⁶ cells) was mixed with the indicated number of cells from strain C699-89 (MATa bar1Δ ste7Δ) with a plasmid expressing the specified Ste7 variant from the STE7 promoter. (B) Pheromone-induced reporter gene expression. The bar graph shows the average β-galactosidase activity for expression of the mating-specific FUS1-LacZ reporter gene (pNC756) in two different strains carrying the specified Ste7 M variant expressed from the STE7 promoter. Averages are from four independent cultures of each variant in strain C699-89 (bar1Δ ste7Δ) before () and after (▪) 2-h induction with 50 nM α-factor and from four independent cultures of each variant in strain MLY218a (MATa ste7Δ) before (□) and after () 2.5-h induction with 3 μM α-factor. Error bars show the average deviations in measurements. (C) Pheromone-induced G₁ arrest and mating differentiation. The bar graph shows the average G₁ arrest and mating differentiation indices for samples from the same cultures used to measure reporter gene expression in panel B. The G₁ arrest index (C699-89 ; MLY218a [□]) is the percentage of unbudded cells in each culture after pheromone induction minus the percentage of unbudded cells in the starting vegetative culture. The differentiation index (C699-89 [▪]; MLY218a []) is the percentage of cells that formed mating projections. Error bars show the average deviations in measurements. Ste7 variants were expressed from the STE7 promoter by using the following plasmids: pNC766 (Ste7M), pNC769 (Ste7M-A₇), pNC793 (Ste7M-E₇), pNC768 (Ste7M-EE), pNC770 (Ste7M-EE-A₇), pNC791 (Ste7M-EE-E₇), pNC767 (Ste7M-AA), and pNC771 (Ste7M-N³⁴⁹).

Invasion assays comparing the in vivo functions of Ste7 phosphorylation site substitution derivatives. (A) Micrographs show invasive colonies from a section of growth chambers inoculated with strain MLY218a (ste7Δ) carrying plasmids for expression of the Ste7 variants as specified in Fig. 4. (B) Bar graph shows the average and average deviation of the invasive colony mean area from three independent experiments. Measurements were made on 25 or more individual colonies in a given chamber after 46 to 48 h of growth at 30°C. Because few invasive colonies formed with the strains expressing the catalytically inactive Ste7M-N³⁴⁹ and Ste7M-AA variants, the values are an average from measurement of only 9 and 11 total colonies, respectively.

Comparison of Fus3 and Kss1 phosphorylation induced by pheromone or constitutive Ste7. (A to C) The upper panel shows the total amount of Fus3M and Kss1M by detection with anti-myc antibodies. The lower panel shows the fraction of dually phosphorylated Fus3M and Kss1M by detection with anti-Erk (pT-E-pY) antibodies. The signals corresponding to Kss1M (K) and Fus3M (F) are indicated to the right of the blots. (A) Protein extracts (33 μg) were from strain C699-140 (STE5 ste7Δ fus3Δ kss1Δ) expressing wild-type (Wt) Ste7 (pNC318), Ste7-EE-A₇ (pNC597), or Ste7-EE-E₇ (pNC893) with Kss1M (pNC977), Fus3M (pNC979), or both, as indicated. Lanes 1 and 2 show extracts from the Wt-Ste7 strain before (−) and after (+) 15-min pheromone (α-fr; 50 nM) induction. (This time was empirically shown to result in optimal induction of Fus3 and Kss1 phosphorylation.) Lane 9 shows an extract from the Ste7-EE-E₇ strain with vectors (pRS313 and pRS316) as a negative reference. (B and C) Lanes 1 and 2 show extracts (66 μg) from strain C699-140 (STE5 ste7Δ fus3Δ kss1Δ) expressing Wt-Ste7 with Kss1M and Fus3M before (−) and after (+) induction with pheromone as specified in panel A. Lanes 3 and 4 (B) or 3 to 5 (C) show extracts (33 μg) from strain C699-90 (ste5Δ ste7Δ fus3Δ kss1Δ) expressing Ste7-EE-A₇ or Ste7-EE-E₇ with Kss1M, Fus3M, or both as specified. Lane 6 (C) shows an extract from strain C699-90 expressing Ste7-EE-A₇ with vectors (pRS313 and pRS316) as a negative reference. (D) Bar graph comparing the relative fold induction of phospho-Kss1 () and phospho-Fus3 () in strains as defined for panels A to C. Values are the average fold induction determined from three experiments. Induction is the fraction of phospho-MAPK [(anti-myc signal)/(anti-pT-E-pY signal)] for each sample relative to that from the uninduced reference. The lines with bars show the average deviations in measurements.

Copurification assays comparing the in vivo binding of Ste7 phosphorylation site substitution derivatives to other components of the MAPK activation module. Representative Western blots show the amount of indicated Ste7 variant and GST fusion protein in the starting extracts (Input) and in the glutathione-agarose-immobilized fraction (Eluant). Proteins were fractionated by SDS-PAGE. Monoclonal anti-myc 9E10 antibodies were used for detection of myc epitope-tagged Ste7 (Ste7M) derivatives, and monoclonal anti-GST antibodies were used for detection of GST or the indicated GST fusion protein. Bar graphs show the average relative binding and average deviation for the specified Ste7M variant and GST fusion protein. Relative binding is the ratio of the anti-myc signal (Ste7) to the anti-GST signal (fusion protein) in the eluant fraction. (A) Strain C699-93 (ste11Δ fus3Δ) was used to coexpress each Ste7M variant with GST-STE5 (pYBS298). Strain C699-92 (ste7Δ ste5Δ ste11Δ fus3Δ kss1Δ) was used to coexpress each Ste7M variant with the following: GST-STE11 (pRD-GST-Ste11^1-717) (B); GST-FUS3 (pNC901) (C); GST-KSS1 (pNC903) (D); or GST (pEG-KT) (E). Indicated Ste7M variant proteins were expressed using the plasmids pNC896 (Ste7M-AA-A₇), pNC897 (Ste7M-EE-A₇), pNC898 (Ste7M-AA-E₇), and pNC809 (Ste7M-EE-E₇).

Invasion assays comparing wild-type and constitutive Ste7 variants in FUS3 and fus3Δ genetic backgrounds. (A) Micrographs show invasive colonies from a section of the invasion chambers inoculated with strain MLY218α (FUS3 ste7Δ) or SM002 (fus3Δ ste7Δ) carrying plasmids for expression of wild-type (Wt) Ste7 (pNC766), Ste7-EE-A₇ (pNC770), Ste7-EE-E₇ (pNC791), or catalytically inactive Ste7-N³⁴⁹ (pNC771) as indicated. (B) Bar graph shows the average and average deviation of the invasive colony mean area from three independent experiments. Measurements were made after 46 to 48 h of growth at 30°C. The number of colonies measured in each experiment depended on the Ste7 variant and FUS3 background. Chambers of the FUS3 strain with functional Ste7 variants contained 29 to 127 invasive colonies; those of the corresponding fus3Δ strain contained 53 to 230 invasive colonies. Chambers of the negative reference (Ste7-N³⁴⁹) strains contained 7 to 27 invasive colonies.

Model for regulation of the mating pathway MAPK cascade. See text for explanation.