Research Article Constitutive activation of the Saccharomyces cerevisiae transcriptional regulator Ste12p by mutations at the amino-terminus Juan Alonso Crosby 1 , James B. Konopka 2 and Stanley Fields 3 * 1 Program in Biochemistry and Molecular Biology, Department of Molecular and Cellular Biology, State University of New York at Stony Brook, Stony Brook, NY 11794, USA 2 Department of Molecular Genetics and Microbiology, State University of New York at Stony Brook, Stony Brook, NY 11794, USA 3 Howard Hughes Medical Institute, Departments of Genetics and Medicine, University of Washington, Box 357360, Seattle, WA 98195, USA * Correspondence to: S. Fields, Howard Hughes Medical Institute, Departments of Genetics and Medicine, University of Washington, Box 357360, Seattle, WA 98195, USA. E-mail: ®elds@u.washington.edu Received: 8 March 2000 Accepted: 1 June 2000 Abstract The transcriptional activator Ste12p is required for the expression of genes induced by mating pheromone in the yeast Saccharomyces cerevisiae. We identi®ed mutations in the amino-terminal DNA-binding domain of Ste12p that lead to constitutively high-level transcription of pheromone-induced genes. The behaviour of these mutant proteins is consistent with an enhanced DNA-binding ability. Cells carrying these hyperactive proteins retain their sensitivity to pheromone treatment, and their phenotype is largely dependent on the presence of at least one of the MAP kinases (Fus3p or Kss1p) and the scaffold protein Ste5p. Deletion of either FUS3 or KSS1 leads to a marked increase in Ste12p activity, consistent with a negative regulatory role for Fus3p, similar to that described for Kss1p. The properties of the constitutive mutants support the idea that the pheromone response pathway plays a role in basal as well as pheromone-induced transcription. Copyright # 2000 John Wiley & Sons, Ltd. Keywords: Ste12p; pheromone response; mating pathway; transcription; MAP kinases; cell-type-speci®c expression Ste12p is a transcriptional regulator essential for the initiation of different developmental programmes in Saccharomyces cerevisiae. In haploid cells, Ste12p regulates the expression of genes essential for mating (Fields and Herskowitz, 1985; Dolan et al., 1989; Errede and Ammerer, 1989). In diploid cells, Ste12p is necessary for the development of ®lamen- tous growth (Liu et al., 1993; Roberts and Fink, 1994). In the mating process, peptide pheromones bind to speci®c receptors on the cell surface (Ste2p in a cells or Ste3p in a cells), resulting in the activation of a signal transduction cascade com- posed of a set of protein kinases (Ste20p, Ste11p, Ste7p, Fus3p and Kss1p). The activity of the kinase cascade is regulated by a heterotrimeric G protein that is coupled to the pheromone receptor. Recep- tor activation produces dissociation of the G protein, with the bc dimer (Ste4p/Ste18p) relaying the signal to the downstream kinases (reviewed in Lebereret al., 1997). The protein kinases interact with the scaffold protein Ste5p, which appears to regulate the speci®city and ef®ciency of the mating signal (Elion, 1995; Mahanty et al., 1999). The activity of the protein kinases may relieve the negative regulatory activity that the Dig1p and Dig2p proteins exert on Ste12p (Cook et al., 1996; Tedford et al., 1997; Bardwell et al., 1998). The MAP kinases, Fus3p and Kss1p, play an additional role in controlling the developmental fate of cells (Sprague, 1998). Kss1p acts as a negative regulator of ®lamentous growth, while Fus3p prevents the inappropriate activation of the ®lamentous growth pathway by pheromone-dependent signalling (Cook et al., 1997; Madhani et al., 1997; Bardwell et al., 1998). The targets of Ste12p include three sets of cell type-speci®c genes: a-speci®c genes (expressed only in a cells), a-speci®c genes (expressed only in a Yeast Yeast 2000; 16: 1365±1375. Copyright # 2000 John Wiley & Sons, Ltd.