Academic journal article Genetics

Snf1-Dependent and Snf1-Independent Pathways of Constitutive ADH2 Expression in Saccharomyces Cerevisiae

Academic journal article Genetics

Snf1-Dependent and Snf1-Independent Pathways of Constitutive ADH2 Expression in Saccharomyces Cerevisiae

Article excerpt

ABSTRACT

The transcription factor Adr1 directly activates the expression of genes encoding enzymes in numerous pathways that are upregulated after the exhaustion of glucose in the yeast Saccharomyces cerevisiae. ADH2, encoding the alcohol dehydrogenase isozyme required for ethanol oxidation, is a highly glucose-repressed, Adr1-dependent gene. Using a genetic screen we isolated >100 mutants in 12 complementation groups that exhibit ADR1-dependent constitutive ADH2 expression on glucose. Temperature-sensitive alleles are present among the new constitutive mutants, indicating that essential genes play a role in ADH2 repression. Among the genes we cloned is MOT1, encoding a represser that inhibits TBP binding to the promoter, thus linking glucose repression with TBP access to chromatin. Two genes encoding proteins involved in vacuolar function, FAB1 and VPS35, and CDC10, encoding a nonessential septin, were also uncovered in the search, suggesting that vacuolar function and the cytoskeleton have previously unknown roles in regulating gene expression. Constitutive activation of ADH2 expression by Adr1 is SNF1-dependent in a strain with a defective MOT1 gene, whereas deletion of SNF1 did not affect constitutive ADH2 expression in the mutants affecting vacuolar or septin function. Thus, the mutant search revealed previously unknown Snf1-dependent and -independent pathways of ADH2 expression.

THE yeast Saccharomyces cerevisiae has a sophisticated system for transducing information regarding nutrient availability to the nucleus (GANCEDO 1998; CARLSON 1999). Glucose, the preferred source of energy, is oxidized to produce carbon dioxide and ethanol during fermentative growth. During this phase of growth the transcription of genes involved in many nonfermentative pathways is repressed, as are genes required for the utilization of less-preferred fermentable substrates, such as galactose, sucrose, and maltose. When glucose is exhausted, these genes are activated to allow the cell to use alternative carbon sources for growth and energy production (SCHULLER 2003), an adaptive change in metabolism that occurs during the diauxic transition. This change in metabolism is accompanied by a massive reprogramming of gene expression (DERiSi etal 1997).

The diauxic transition is regulated by the Snfl protein kinase, the yeast homolog of the mammalian AMPactivated protein kinase (AMPK) (HARDIE et al. 1998). Snfl is part of a kinase complex whose activity is stimulated by low glucose concentration. The activity of the Snfl kinase complex is regulated by Glc7.Regl.Bmh, a type I protein phosphatase complex (SANZ et al. 2000; DOMBEK et al. 2004) ; three targeting subunits ( SCHMIDT and McCARTNEY 2000); and three upstream kinases (HONG et al. 2003). Many of the genes whose expression is Snfl-dependent encode regulatory proteins, such as protein kinases, protein phosphatases, and transcription factors, suggesting that Snfl acts through a complex regulatory cascade (YouNG et al. 2003).

Adrl and Cat8 are two transcription factors that act downstream of Snfl to activate nonfermentative metabolic pathways (SCHULLER 2003). Adrl and Cat8 act both independently and synergistically to regulate > 1OO genes after the diauxic transition (YouNG et al. 2003; TACHIBANA et al. 2005). One of the Snfl-dependent genes activated by Adrl and Cat8 is ADH2, encoding alcohol dehydrogenase II, the isozyme that catalyzes the first step in ethanol oxidation. No DNA-binding repressers of ADH2 transcription have been identified (!RANI et al. 1987). Instead, ADH2 expression is repressed by the absence of active Snfl, which is kept in an inactive state in the presence of glucose by an active Glc7.Regl.Bmh complex (DOMBEK et al. 1993, 2004). Activation (derepression) of ADH2 expression requires the cooperative binding of Adrl and Cat8, leading to synergistic activation when both factors are present (WALTHER and SCHULLER 2001; TACHIBANA et al. 2005). Snfl regulates both the expression and the activity of Cat8 (RAHNER et al. …

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