Isolation of Suppressors of the Inhibition of the Pheromone Response Pathway Posed by Overexpression of the Ste11 Amino Terminus.

Pheromone response employs a mitogen activated protein kinase (MAPK) signal transduction pathway composed of Ste20 (MAPKKKK), Ste11 (MAPKKK), Ste7 (MAPKK), and Fus3/Kss1 (MAPK). The mechanism for activation of the Ste11 kinase is not well understood. Deletion of the Ste11 N-terminal 352 amino acids results in constitutive pathway activation, suggesting that this region is a negative regulatory domain. We find that overexpression of the N-terminal 209 amino acids inhibits signal output required for both transcriptional activation and G1 arrest responses to pheromone. We conducted a screen to search for proteins which when overexpressed would relieve this Ste11 N-terminus-mediated inhibition created by integration of GALSTE11^1-209. The pheromone-inducible FUS1 promoter linked to the HIS3 gene was integrated at the HIS3 locus, necessitating restoration of signal output for growth of this strain on media lacking histidine. We screened 20,000 transformants from a genomic overexpression library (pYES-YGL) and isolated 4 different clones that allowed growth on -his Gal medium. These clones express Ste4, Ste11^296-717, Ash1, and Ste12^96-688 . Although not recovered in our screen, overexpression of Ste5 was also shown to reverse the inhibition of signaling caused by Ste11^1-209. The different mechanisms by which these various gene products relieve the Ste11 N-terminus-mediated inhibition of signal transduction will be discussed.