The Histone H2B Arg95 Residue Recruits Spt16 to Maintain a Proper Expression of STE5 Required for Transmitting Rapamycin-Induced G1 Arrest in Saccharomyces cerevisiae

The immunosuppressant rapamycin inhibits the TORC1 kinase pathway in yeast, leading to cell cycle arrest through a mechanism that is not fully understood. To better understand this process, we used a collection of 442 histone mutants and subjected these to different concentrations of rapamycin in order to isolate histone mutants that are particularly resistant to rapamycin. We recently reported a histone mutant, H2B R95A, that is strikingly resistant to rapamycin and which we characterized to be defective in the expression of nearly 25 genes belong to the pheromone pathway like STE5 encoding a scaffold protein that recruits MAP kinases for activation. Mutants lacking Ste5 were resistant to rapamycin and expressed ~ 3- to 4-fold higher levels of the G1 cyclin Cln2 as compared to the parent, suggesting that the pheromone pathway transmits the rapamycin signal. We also investigated how H2B R95A prevents the expression of the STE5 gene. We discovered that H2B R95A mutant is deficient in recruiting the histone chaperone Spt16 as compared to the H2B WT. We showed that Spt16 mutants, spt16-E857K and spt16-11, were sensitive to rapamycin, which correlated with the expression of different forms of Ste5, suggesting that these alternative forms of Ste5 might cause toxicity upon rapamycin exposure. We postulate that the rapamycin-sensitive Spt16 mutants expressed Ste5 forms that prevent the induction of the Cln2 cyclin. Our findings suggest new insights into how rapamycin signaling, facilitated by the Torc1 kinase, may utilize the pheromone pathway to halt cells in the G1 phase.