Biochemical and genetic analysis of a novel gene STD1: A transcriptional regulator in yeast

摘要

The STD1 gene was isolated as a high-copy-number suppressor of the growth defect of yeast strains engineered to overexpress a mutant TATA binding protein (TBP). The selective defects in uninduced and induced RNA pol II transcription caused by mutant TBP overexpression were corrected by Std1 overexpression. STD1 (MSN3) was also cloned in the Carlson lab as a high-copy-number suppressor of the growth defect of $snf4Delta 2$ mutants growing upon raffinose media. STD1 was shown to be a gene-dosage dependent modulator of glucose repression of SUC2 transcription. Std1 can interact with both the TBP and with the Snf1 kinase as demonstrated in vivo with the yeast two hybrid system, and in vitro with co-immunoprecipitation and column binding assays. The data suggest that Std1 couples the glucose starvation signal from the Snf1 kinase complex to TBP in regulating transcription by glucose. Utilizing Std1 in the two-hybrid system, the C-terminal tails of the glucose sensors Snf3 and Rgt2 have been cloned and a variety of data indicate that Std1 interactions with Snf3 and Rgt2 are important in regulating transcription by glucose.;It was discovered that mutations in STD1, SNF3, RGT2, SNF4, and SNF5 affect growth in the presence of sodium stress indicating a connection in the pathways responding to glucose and sodium. The basis of this connection is partially reflected in the glucose and sodium-regulated expression of HAL1 mRNA. Hal1 is involved in retaining intracellular potassium, which is an important response to sodium stress. Overexpression of Std1 or deletion of Snf3 and Rgt2 increases HAL1 mRNA expression, whereas deletion of Stdl and Mth1 or Snf5 decreases HAL1 mRNA expression. The conserved protein phosphatase 2B (calcineurin) is required in the response to sodium stress and affects the sodium-induced transcription of ENA1, encoding an effluxive sodium pump. Both Stdl and calcineurin mutants are defective for growth in the presence of Na$sp+$, Li$sp+$, Mn$sp{++}$, or OH$sp-$ ion stress. Std1 overexpression can suppress calcineurin defects, suggesting that Std1 functions downstream of or in parallel to calcineurin. Std1 functions in the glucose derepression pathway and is important in the maintenance of ion homeostasis.