Date of Award

Fall 1988

Project Type


Program or Major


Degree Name

Doctor of Philosophy


The ADR1 protein activates transcription of the gene coding for the glucose repressible alcohol dehydrogenase II (ADH2) in Saccharomyces cerevisiae. Previous characterization of ADR1$\sp{\rm c}$ mutations, which allow ADH2 to escape glucose repression, led to the hypothesis that ADR1 is inactivated post-translationally by a cAMP-dependent phosphorylation mechanism. This hypothesis was tested by investigating the in vitro phosphorylation of ADR1 and ADR1$\sp{\rm c}$ proteins in which ADR1$\sp{\rm c}$ proteins were predicted to display a diminished level of phosphorylation. A fragment of the ADR1 gene coding for the N-terminal 658 amino acids was fused to the lacZ gene of E. coli to allow for identification and assay of the ADR1 protein. The fused gene was placed under the control of the E. coli tac promotor to ensure efficient production. Beta-galactosidase activities in plasmid-bearing E. coli indicated that ADR1/$\beta$-galactosidase fusion proteins were being expressed. SDS-polyacrylamide gel analysis of E. coli extracts revealed discrete plasmid-encoded fusion proteins.

Fusion proteins contained in crude E. coli extracts were phosphorylated in vitro using purified catalytic subunit of mammalian cAMP-dependent protein kinase (cAPK). Peptide mapping results indicated that ADR1 was phosphorylated at two discrete sites in vitro. The primary phosphorylation site was identified as serine-230. A secondary site to the N-terminal side of serine-230 was also identified.

ADR1 DNA sequences were replaced in the expression plasmids with three sequences containing ADR1$\sp{\rm c}$ mutations, each of which encodes a single amino acid substitution within the phosphorylation sequence located between residues 227-231 (Arg-Arg-Ala-Ser-Phe). The in vitro phosphorylation of ADR1 and ADR1$\sp{\rm c}$ containing fusion proteins were compared using both bovine cAPK and the yeast cAPK catalytic subunit encoded by the TPK1 gene. Addition of purified yeast regulatory subunit completely blocked fusion protein phosphorylation by the yeast kinase in the absence of cAMP. The ADR1-2$\sp{\rm c}$ and ADR1-5$\sp{\rm c}$ mutations were found to diminish phosphorylation at serine-230. The ADR1-7$\sp{\rm c}$ mutation, which substitutes a leucine residue for serine-230, completely eliminated phosphorylation.

The results of this work support the hypotheses that the ADR1$\sp{\rm c}$ proteins bypass glucose repression by avoiding phosphorylation and suggest that ADR1 is regulated by a cAMP-dependent phosphorylation. Possible mechanisms by which phosphorylation may regulate ADR1 activity are discussed.