Overexpression of transcriptional factors Kin28 and Pog1 suppresses the stress sensitivity caused by the rsp5 mutation

Rsp5 is an essential and multi-functional E3 ubiquitin ligase in Saccharomyces cerevisiae. We previously isolated the Ala401Glu rsp5 mutant, which is hypersensitive to various stresses. To understand the function of Rsp5 in stress responses, suppressor genes whose overexpression allows rsp5A401E cells to grow at high temperature were screened. The KIN28 and POG1 genes, encoding a subunit of the transcription factor TFIIH and a putative transcriptional activator, respectively, were identified as multicopy suppressors of not only high temperature but also LiCl stresses. The overexpression of Kin28 and Pog1 in rsp5A401E cells caused an increase in the transcriptional level of some stress proteins when exposed to temperature up-shift. DNA microarray analysis under LiCl stress revealed that the transcriptional level of some proteasome components was increased in rsp5A401E cells overexpressing Kin28 or Pog1. These results suggest that the overexpression of Kin28 and Pog1 enhances the protein refolding and degradation pathways in rsp5A401E cells. Keywords: mutant analysis, stress response Total RNA from S. cerevisiae was isolated by the method of Köhrer and Domdey (1991). Poly A mRNA was enriched from total RNA by Oligotex dT30 mRNA purification kit (Takara Bio). The Affimetrix yeast genome S98 arrays (YGS98 GeneChip, Affymetrix, Santa Clara, CA) were used as DNA microarray in this study. The biotinyated cRNA (15 μg) probe was hybridized to DNA microarray at 45°C for 18 h according to Affymetrix user’s manual. The washing and staining of arrays were performed using the GeneChip Fluidics Station 400. The scanning of arrays was carried out using the GeneArray scanner (Agilent technologies, Palto Alto, CA).