Regulation of weak acid stress responses and ribosomal protein gene expression in Saccharomyces cerevisiae

Abstract

Saccharomyces cerevisiae is widely used in molecular and cell biology since it is one of the simplest eukaryotic single cell. Due to its easiness in manipulation and fast growth, S. cerevisiae has served as a model system for fundamental cellular processes for all eukaryotes. In nutrient rich conditions, yeast cells rapidly grow and proliferate. To maintain robust growth, yeast cells generate numerous ribosomes to synthesize proteins in need. In response to stresses, cells rapidly adjust global gene expressions to adapt to stresses. For rapid growth and stress responses, cells activate transcription factors to regulate gene expressions. In this dissertation, regulatory mechanisms of transcription factors Haa1/War1 for weak organic acid stresses and Ifh1/Crf1 for the expression of ribosomal protein genes were studied.<br/> Firstly, S. cerevisiae is known to activate transcription factors such as Haa1 and War1 for cellular adaption against weak acids. Haa1 plays important roles against less lipophilic acids such as acetic acid and lactic acid, whereas War1 exert protection against more lipophilic acids such as propionic acid, sorbic acid, and benzoic acid. However, it has been unknown how these transcription factor are activated in response to weak acid stresses. Using field-effect transistor (FET) type biosensor based on carbon nanofibers, it has been revealed that Haa1 and War1 directly bind to weak acid anions with varying affinities. In agreement with previous studies, Haa1 binds acetate most strongly followed by lactate, whereas War1 binds benzoate most strongly followed by sorbate. Upon activation of Haa1 by direct binding with acetate, Haa1 binds to DNA of target gene promoters through the N-terminal Zn-binding domain of Haa1. Acetate is shown to bind N-terminal 150 amino acid region of Haa1, however, further C-terminal region of Haa1 is required for acetic acid induced transcriptional activation of its target genes. Therefore, it is proposed that conformation changes caused by direct binding of acetate may activate Haa1, being capable of DNA binding and transcriptional activation.<br/> Secondly, transcription factors Ifh1 and Crf1 are involved in the regulation of ribosomal protein (RP) genes through interaction with a forkhead-associated (FHA) domain containing transcription regulator Fhl1. The FHA domain of Fhl1 interacts with FHB domains of RP gene co-activator Ifh1 or co-repressor Crf1. Ifh1T681 and Crf1T348, which are resides in the FHB domain, is phosphorylated by CK2 kinase. These phosphorylations play very critical roles for interaction with Fhl1. Cells expressing Ifh1T681A mutant showed reduced phosphorylation by CK2 followed by substantially decreased interaction with Fhl1. Decreased interaction resulted in defects in association of Ifh1 at the RP gene promoters and finally reduced RP gene transcriptional activation, thereby resulting in slow growth rates. On the contrary, cells expressing Crf1T348A failed to repress RP gene transcription upon inhibition of target of rapamycin complex 1 (TORC1) by rapamycin treatment. Taken together, these results propose that CK2-dependent phosphorylation of transcription factors, Ifh1 and Crf1, regulates recruitment transcription factors at the RP gene promoters, which leads to transcription of RP genes. <br/>