Regulation of expression and activity of the yeast transcription factor ADR1.

Disruption of ADR1, a positive regulatory gene in the yeast Saccharomyces cerevisiae, abolished derepression of ADH2 but did not affect glucose repression of ADH2 or cell viability. The ADR1 mRNA was 5 kilobases long and had an unusually long leader containing 509 nucleotides. ADR1 mRNA levels were regulated by the carbon source in a strain-dependent fashion. beta-Galactosidase levels measured in strains carrying an ADR1-lacZ gene fusion paralleled ADR1 and ADR1-lacZ mRNA levels, indicating a lack of translational regulation of ADR1 mRNA. ADH2 was regulated by the carbon source to the same extent in all strains examined and showed complete dependence on ADR1 as well. The expression of ADR1 mRNA and an ADR1-beta-galactosidase fusion protein during glucose repression suggested that the activity of the ADR1 protein is regulated at the posttranslational level to properly regulate ADH2 expression. The ADR1-beta-galactosidase fusion protein was able to activate ADH2 expression during glucose repression but showed significantly higher levels of activation upon derepression. A similar result was obtained when ADR1 was present on a multicopy plasmid. These results suggest that low-level expression of ADR1 is required to maintain glucose repression of ADH2 and are consistent with the hypothesis that ADR1 is regulated at the posttranslational level.     

The Reg1-interacting proteins, Bmh1, Bmh2, Ssb1, and Ssb2, have roles in maintaining glucose repression in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, a type 1 protein phosphatase complex composed of the Glc7 catalytic subunit and the Reg1 regulatory subunit represses expression of many glucose-regulated genes. Here we show that the Reg1-interacting proteins Bmh1, Bmh2, Ssb1, and Ssb2 have roles in glucose repression. Deleting both BMH genes causes partially constitutive ADH2 expression without significantly increasing the level of Adr1 protein, the major activator of ADH2 expression. Adr1 and Bcy1, the regulatory subunit of cAMP-dependent protein kinase, are both required for this effect indicating that constitutive expression in Deltabmh1Deltabmh2 cells uses the same activation pathway that operates in Deltareg1 cells. Deletion of both BMH genes and REG1 causes a synergistic relief from repression, suggesting that Bmh proteins also act independently of Reg1 during glucose repression. A two-hybrid interaction with the Bmh proteins was mapped to amino acids 187-232, a region of Reg1 that is conserved in different classes of fungi. Deleting this region partially releases SUC2 from glucose repression. This indicates a role for the Reg1-Bmh interaction in glucose repression and also suggests a broad role for Bmh proteins in this process. An in vivo Reg1-Bmh interaction was confirmed by copurification of Bmh proteins with HA(3)-TAP-tagged Reg1. The nonconventional heat shock proteins Ssb1 and Ssb2 are also copurified with HA(3)-TAP-tagged Reg1. Deletion of both SSB genes modestly decreases repression of ADH2 expression in the presence of glucose, suggesting that Ssb proteins, perhaps through their interaction with Reg1, play a minor role in glucose repression.     

Identification and Characterization of Three Genes That Affect Expression of Adh2 in Saccharomyces Cerevisiae

Using a new selection protocol we have identified and preliminarily characterized three new loci (ADR7, ADR8 and ADR9) which affect ADH2 (alcohol dehydrogenase isozyme II) expression. Mutants were selected which activate ADH2 expression in the presence of an over-expressed, normally inactive ADR1 allele. The mutants had very similar phenotypes with the exception that one was temperature sensitive for growth. In the absence of any ADR1 allele, the mutants allowed ADH2 to partially escape glucose repression. However, unlike wildtype strains deleted for ADR1, the mutants were able to efficiently derepress ADH2. The mutations allowed a small escape from glucose repression for secreted invertase, but had no effect on the glucose repression of isocitrate lyase or malate dehydrogenase. The mutations were shown to be nonallelic to a wide variety of previously characterized mutations, including mutations that affect other glucose-repressed enzymes.