ADH2 repression: A genetic and biochemical approach

In the yeast Saccharomyces cerevisiae, glucose represses the expression of ADH2 (alcohol dehydrogenase II) the enzyme that catalyzes the first step in the utilization of ethanol as a carbon source for non-fermentative growth. Whereas we know the essential elements responsible for ADH2 activation, which are the Snf1 kinase complex, DNA binding activators Adr1 and Cat8, our understanding of the mechanism of glucose repression is less complete. Several trans-acting mutations are known that partially decrease the stringency of glucose repression, including three mutations in three genes of unknown function adr7, adr8 and adr9. We cloned the ADR7 gene and demonstrated that it is identical to MOT1, a general repressor of RNA pol II transcription. We found also that Mot1 interacts genetically with a particular sequence in the ADH2 promoter region. A double adr7ADH2-10 mutant that has both adr7/mot1 mutation and a ten bp deletion in the promoter region of ADH2 exhibits a synergistic effect on the level of constitutive ADH2 activity. Investigating the genetic interaction between the adr7/ mot1 mutant and an adr8 mutant, we found that adr7adr8 double mutants are synthetically lethal. This observation suggests as one possibility that Adr7/Mot1 and Adr8 are part of two pathways that overlap in function and, therefore, when Adr7/Mot1 is defective the function of Adr8 becomes essential for cell viability. Our attempts to clone ADR8 failed, but we succeeded in cloning a multi-copy suppressor of the adr8 mutant, RSP5. RSP5 encodes an essential HECT E3 ubiquitin-protein ligase and plays an essential role in ubiquitin-mediated endocytosis and subsequent vacuolar degradation of membrane proteins as well as nuclear proteins, such as the Rpb1 (the largest subunit of pol II). We showed that the level of the activator Adr1₂₂₀ is significantly reduced in the presence of a multi-copy plasmid pVC-8 ( RSP5) in the adr8 mutant. This result made us realize that a reduced level of the Adr1 activator under repressed conditions could be an obstacle for detecting constitutive ADH2 mutants.; Thus, using a strain with additional copies of ADR1 we used a genetic screen to isolate mutants representing more than twelve complementation groups that are defective in ADH2 repression by glucose. Among these complementation groups we found genes known to affect ADH2 repression, such as ADR7/MOT1, ADR8 and GLC7 as well as previously unknown genes including two genes encoding proteins involved in the vacuole protein sorting pathway: FAB1 (PtdIns(3′)P-5′-kinase) and VPS35 (part of the retromer complex).