Phosphorylation of Adr1 phosphorylation at the serine within the sequence RTPSGK at amino acids 95–100. To determine if serine 98 was phosphorylated in vivo, we used antibodies raised against LRLNGRTP(pS)GKLRSFVC, where (pS) is phosphorylated serine. Adr1 is difficult to detect in repressed conditions, so we used strains containing a multi-copy plasmid with ADR1 under control of the constitutive ADH1 promoter. To study the specificity of the antiserum and the effect of point mutants that mimic the non-phosphorylated and phosphorylated state, serine 98 on Adr1 was changed to alanine and aspartate in related plasmids, referred to as S98A and S98D. The anti-phosphoserine 98 peptide antiserum (a-pS98) recognized a protein in yeast cell extracts that co-migrated with Adr1, was absent in cells lacking Adr1 (data not shown), and was greatly reduced when Adr1-S98A or Adr1-S98D was expressed instead of wild-type Adr1 (Figure 2A). The specificity of a-pS98 was confirmed when the phosphorylated peptide efficiently competed with the protein detected on the western blot, but a non- phosphorylated synonymous peptide did not (Figure 2B). In summary, the serine 98 proximal to the Zn-finger domain of Adr1 is phosphorylated both in vitro and in vivo. Adr1-Ser98 phosphorylation is affected by glucose repression and SNF1 If phosphorylation of Adr1 at serine 98 is relevant to its activity in vivo, we would expect the level of phosphorylation to change in glucose derepressing conditions. We measured serine 98 phos- phorylation in extracts from cells shifted to low glucose by western blotting with anti-pS98 and anti-Adr1 antiserum. The intensity of the anti-pS98 signal of each sample was normalized to its signal with anti-Adr1. We found that derepression was accompanied by a decrease in serine 98 phosphorylation relative to total Adr1 of five- fold (average of three separate experiments, standard deviation of two, Figure 2A). Glucose regulation of Adr1 occurs through SNF1 because Adr1 does not bind promoters in a Dsnf1 strain [7]. To test the effect of Snf1 on Adr1 serine 98 phosphorylation, we used strains with four integrated copies of ADR1 expressed from its own promoter, one of which was tagged with a triple HA epitope. Glucose repression is maintained in this strain despite a modestly elevated level of Adr1 [6]. Figure 2C shows Adr1 serine 98 phosphorylation in a strain lacking Snf1. The decrease in phosphorylation relative to total Adr1 in low glucose was less than two-fold (average of two experiments, standard deviation of Figure 2. In vivo phosphorylation of Adr1 at serine 98. A. Western blots with antibody against phosphorylated serine 98 of Adr1 (a-pS98) or total Adr1 (a-Adr1 or a-HA) were performed on 50 mg of cell extract per lane from Dadr1 strain TYY497 with high-copy plasmid pNKA1 (WT, wildtype Adr1), pNKA2 (Adr1-S98D) or pNKA4 (Adr1-S98A). Derepressed cells were grown in 0.05% glucose for one hour. B. Western blots were performed as in A with a-pS98 or a-HA epitope antibody. Each lane contains 20 mg of repressed cell extract from TYY497 with plasmid pKD17-HA. Competing phosphorylated (pS98)-, or non-phosphorylated Ser98 (S98)-containing peptides were added to the primary antibody at 10 mg/ml. C. Western blots were performed as in B on 50 mg per lane of NK91. Derepressed cells were grown in 0.05% glucose, 2% ethanol for one hour. In this section, the lower panel shows the merged images of a-pS98 and a-HA. doi:10.1371/journal.pone.0003213.g002 PLoS ONE | www.plosone.org 3 September 2008 | Volume 3 | Issue 9 | e3213