Table 3. Beta-galactosidase assays with ADH2 promoter-lacZ in Dpho80, Dpho85, Dpcl10 straina WT Dpho85 Dpho80 Dpcl10 ß-galactosidase activity (Miller Units) 5.3 (1.7)b 180 (103) 28 (1.1) 2.6 (0.8) aNK85, NK135, NK136 or NK139 grown in YPD 5% glucose bNumbers in parentheses are standard deviations for the average of duplicate cultures doi:10.1371/journal.pone.0003213.t003 that had been negative in the initial screen, on transcription activation by Adr1. Strains containing an ADH2 promoter/lacZ reporter and four integrated copies of ADR1 [6], were deleted for PHO85 or Pho85-associated cyclin genes. PHO85 and PHO80, but not PCL6 or PCL10 affected ADH2-lacZ expression by beta- galactosidase assays in repressed conditions and blue/white color on X-gal-glucose plates (Table 3 and data not shown). To test for direct phosphorylation of the Adr1-DBD by Pho80/ 85, in vitro kinase assays [29] were performed with purified Pho80/ 85 complex (a gift from Ian Carter-O’Connell from Erin O’Shea’s lab) on purified recombinant Adr1 DBD, or the unphosphorylated 17-amino acid serine 98-containing peptide that was generated for affinity purification of a-pS98. We could not detect phosphory- lation of the Adr1 DBD with the purified kinase-cyclin. We assume the effect of PHO85 on Adr1 phosphorylation that is seen in Figure 6 is indirect, since purified Pho80/85 could phosphorylate a purified Pho4 control, but did not phosphorylate the Adr1 DBD or the serine 98-containing peptide (data not shown). Discussion Serine 98 of Adr1 is involved in regulation of DNA- binding Adr1 directly regulates at least 30 glucose-repressed genes [4,21] and is itself regulated at the level of DNA-binding [7]. We found that mutation of serine 98 in the PAR region of Adr1, which is crucial for optimal binding [15,16,20,30], had a severe effect on in vivo binding to, and activation of the ADH2 promoter. Mutation to the phosphomimetic amino acid aspartate had more deleterious effects than mutation to alanine on both in vitro binding and activation, suggesting that phosphorylation of serine 98 might have similar consequences. The effect of the alanine mutation on Adr1- dependent genes ranged from 5-fold or greater decreases in binding and activation (ADH2), to no effect (ADY2). This may reflect differences in the structure and coactivator requirements of the different promoters, which we find leads to measurable differences in the DNA binding or activator function of Adr1 [31]. Serine 98 is adjacent to the proline at position 97 that creates a turn in the structure of the DBD [16], which may explain why even mutation to a neutral amino acid affects activator function. Serine 98 was phosphorylated in vivo, and phosphorylation was affected by both glucose availability and Snf1. SNF1 was required for the decrease in phosphorylation in response to derepressing conditions. Several possible mechanisms could explain how post-transla- tional modification of Adr1 may regulate promoter binding and transcription activation. Adr1 promoter-binding may be stabilized by interactions with coactivators [32], and post-translational modification may regulate this interaction. A plausible hypothesis Phosphorylation of Adr1 is that phosphorylation interferes with coactivator interaction. However, the close proximity of serine 98 to the DBD of Adr1 (Figure 7) decreases the likelihood of direct coactivator binding. In addition, the Adr1 DBD binds DNA in absence of coactivators in the in vitro EMSA assay, suggesting a reasonably stable binding without assistance. Another candidate for regulating interaction with stabilizing coactivators is serine 230 on Adr1. This residue also appears to be phosphorylated, with relative levels of phosphorylation changing in derepression (S. Ratnakumar and N. Kacherovsky, unpublished). Since this residue is far from the DNA-binding domain, it is a better candidate for regulating interactions with coactivators. Since serine 98 is in a region known to be important for DNA-binding, and mutation affects binding in vivo and in vitro, we propose that phosphorylation of this residue directly affects Adr1-DNA interactions. A model of the Adr1 DBD binding to DNA based on nuclear magnetic resonance spectros- copy of the complex in solution, shows the serine 98 in a position to influence interactions between the PAR domain and DNA. (Figure 7). Since mutation of serine 98 affects binding in vivo and in vitro, our current working hypothesis is that phosphorylation of this residue affects Adr1-DNA interactions. In vivo evidence suggested the Pho80/85 kinase/cyclin complex contributed to phosphorylation of Adr1 at serine 98; however, in vitro evidence suggested the effect was indirect, and serine 98 is not within a Pho85 consensus sequence [33]. We had observed that the Dpho85 strain with the high-copy ADR1 plasmid grew approximately 75% slower than a PHO85 strain carrying the same plasmid, suggesting a physiologically relevant interaction between Pho85 and Adr1. The slower growth rate was partially because of the pho85 mutation, but the growth rate of the Dpho85 strain with the high-copy ADR1-S98D plasmid was only 19% slower than the comparable PHO85 strain. The synthetic deleterious effect between high-copy ADR1 and Dpho85, with partial rescue by mutation of the phosphorylatable residue on Adr1 suggests a common pathway. Pho85 involvement in Adr1 regulation was also observed in a gene expression microarray performed in a Dpho85 strain undergoing the diauxic transition [24]. Many Adr1-dependent genes were characterized as having elevated expression in the pho85 mutant before the cells had exhausted their supply of glucose. Pho85, paired with the cyclin subunits Pcl8 and Pcl10, is involved in glycogen synthesis, in the same pathway as Snf1 [34], and a pho85 mutant has growth defects on non-glucose carbon sources [35]. Here we present data that support a function for Pho85 in glucose repression. Materials and Methods Strains and plasmids Yeast strains are listed in Table 1. Epitope-tagging was as described in Knop et al. [36] and gene deletion was as described in Gu¨ ldener et al. [37]. Cells were grown as described in Sherman [38]. For glucose repression, medium contained 5% glucose; for derepression, 0.05% glucose with or without 2% ethanol. Plasmid pF1F2 (Adr1 17–160 a.a.) [15] was used to express the Adr1 DBD in E. coli for purification. Plasmids F1F2S98A and F1F2S98D were generated from pF1F2 using the Stratagene QuickChange kit. pNKA1, pNKA2, pNKA4, which have the genes for wildtype, S98D and S98A Adr1 DBD, respectively, were made by GAP repair [39] using PCR fragments generated from pF1F2, pF1F2S98D, and pF1F2S98A, cotransformed into yeast with linearized pKD46, which has ADR1 under control of the ADH1 promoter, on a 2m-TRP1 plasmid (K. Dombek, unpub- lished). pNKA1-U is pNKA1 with the TRP1 marker swapped for PLoS ONE | www.plosone.org 6 September 2008 | Volume 3 | Issue 9 | e3213