0.4), so SNF1 was required for the full decrease in serine 98 phosphorylation normally seen in low glucose. Adr1-serine98 mutants have reduced DNA binding affinity in vitro and in vivo Serine 98 phosphorylation decreases in low glucose, while Adr1 promoter-binding increases, and both processes are Snf1-depen- dent ([7], Figure 2). Therefore, we hypothesized that the DNA- binding of Adr1 would be affected by its serine 98 phosphorylation state. In support of this hypothesis, serine 98 is in a region previously shown to be important for DNA binding, the PAR adjacent to the Zn-finger domain [15,16,20]. The DNA binding affinity for recombinant proteins from the wild-type, S98A and S98D plasmids were measured using an electrophoretic mobility shift assay (EMSA). Figure 3 shows that the neutral S98A mutation did not affect in vitro DNA binding, but the phosphomimetic S98D mutation reduced DNA binding affinity approximately 10-fold. The apparent equilibrium constant of Adr1-S98D was 200nM compared to about 20nM for the wild-type and S98A mutant proteins. Other mutations in PAR, such as at proline 97, similarly reduced DNA binding affinity in vitro [20]. Chromatin immunoprecipitation (ChIP) analysis with quantita- tion by real-time PCR (qPCR) was used to compare in vivo promoter-binding of wild-type Adr1 and the S98 mutants. As expected, binding of the Adr1-S98D mutant was at or below background levels, even when over-expressed from a 2m plasmid Phosphorylation of Adr1 under ADH1 promoter-control (data not shown). The Adr1-S98A mutant, however, gave unexpected results. When expressed from a CEN plasmid to ensure physiological levels, binding of the S98A mutant was reduced strongly at ADH2 compared to wildtype Adr1 (Figure 4). Binding was also assayed at POX1, ATO3 and ADY2, which are all highly glucose-repressed, strongly Adr1-dependent for expression, and have promoters that are bound by Adr1. ADH2, ADY2 and ATO3 are Snf1-dependent, but POX1 is not [4,21]. Binding of the S98A mutant was as strongly affected at POX1 as it was at ADH2, and was significantly decreased at ATO3. In summary, mutation of Adr1 serine 98 to the phosphomimetic amino acid aspartate reduced DNA binding affinity in vitro and led to loss of in vivo binding detection. Mutation to alanine did not affect in vitro DNA binding, but reduced in vivo binding. Mutation of Adr1-Ser98 to Asp or Ala decreases transcription activation Since mutation of serine 98 affected the ability of Adr1 to bind promoters, we tested the effect on activation of glucose-repressed genes. First, a yeast strain lacking Adr1 was transformed with multi- copy plasmids expressing wild-type Adr1, Adr1-S98A or Adr1-S98D under the control of the ADH1 promoter. The activity of over- expressed Adr1 was assayed using an Adr1-responsive integrated ADH2/lacZ reporter gene. Wild-type Adr1 was active in both repressing and derepressing medium as expected, because over- expression of Adr1 to this level overcomes glucose repression ([22,23], Figure 3. In vitro binding of Adr1DBD and Ser98 mutants. A. Electrophoretic mobility shift assays (EMSA) were performed with increasing amounts of Adr1 DBD (amino acids 17–160) and its Ser98Ala (S98A) and Ser98Asp (S98D) variants, against a fluorescently labeled double-stranded oligonucleotide containing the Adr1 binding site (UAS1) from the ADH2 promoter. Blue line, wildtype; red line S98A; green line S98D. Error bars represent the standard deviation of the mean measured in duplicate experiments. B. EMSA gel showing mobility shifts of the ADH2 promoter probe with wildtype Adr1 DBD or the S98D mutant. doi:10.1371/journal.pone.0003213.g003 PLoS ONE | www.plosone.org 4 September 2008 | Volume 3 | Issue 9 | e3213