Ssb2 is a novel factor in regulating synthesis and degradation of Gcn4 in Saccharomyces cerevisiae

Summary Yeast cells respond to environmental stress by inducing the master regulator Gcn4 to control genes involved in biosynthesis of amino acids and purine pathways. Gcn4 is a member of the basic leucine Zipper family and binds directly as a homodimer to a conserved regulatory region of target genes. Ssb2 was discovered to rescue the mutant Gcn4 which has a point mutation that decreases DNA‐binding affinity. Ssb2 is part of the Hsp70 protein family responsible for protein quality control and it is thought that Ssb2 assists the passage of nascent polypeptide chains from the ribosomes. To characterize the mechanism behind the rescue of the mutant gcn4 phenotype, transcriptional activity and protein levels of Gcn4 were analyzed. We found that Ssb2 improved the expression of Gcn4 target genes by increasing the DNA‐binding affinity of gcn4 mutants to target gene promoters under conditions of amino acid starvation. Gcn4 levels increased at both translational and post‐translational levels without regulating GCN4 steady‐state mRNA levels. We also found that the nuclear export signal of Ssb2 is required for interaction with Gcn4 and rescue of the gcn4 mutant phenotype. These findings suggest that Ssb2 is a critical factor that modulates Gcn4 functions in the nucleus and cytosol. (A) Wild‐type Gcn4 is recruited to a target gene in the nucleus under starvation conditions and the transcription levels of Gcn4 target genes increased. In case of overexpression of Ssb2, the stability of wild‐type Gcn4 is increased by an interaction with NES of Ssb2 containing RAC or Sis1 in the cytosol. (B) Mutant Gcn4 is not recruited to a target gene in the nucleus under starvation conditions and the transcription of a Gcn4 target gene is not occurred. When Ssb2 is overexpressed, mutant Gcn4 is stabilized by interaction with NES of Ssb2 containing RAC or Sis1 in the cytosol and recruited to a target gene in the nucleus. Thus, transcription levels of Gcn4 target genes are increased.