Phosphorylation of Adr1 Figure 4. In vivo binding of Adr1 and S98A mutant. Chromatin immunoprecipitation assays were performed using anti-HA antibody on the Dadr1 strain TYY497 with pKD16-HA (wildtype, HA-tagged Adr1, black bars) and pKD16-9-HA (Ser98Ala, HA-tagged Adr1, gray bars) derepressed in 0.05% glucose for 3 hours. Quantitation was performed by RTqPCR for the indicated promoters normalized to a non-Adr1- bound telomeric region. Error bars are the standard deviation for the average of technical replicates. doi:10.1371/journal.pone.0003213.g004 Table 2). Despite the high level of expression, the S98D mutant had very low activity in both repressing and derepressing conditions. The S98A mutant was slightly less active than the wild-type protein. Western blots for Adr1-HA protein showed similar levels of wild-type and mutant proteins, indicating that the inactivity of Adr1-S98D was not due to poor expression or instability (data not shown). Some ADH2 expression was seen in a strain with the Adr1-S98A mutant, in contrast to the Adr1-S98D mutant strain, which had less than 10% of wildtype expression levels. To examine the Adr1- S98A mutant at physiological levels, the S98A mutant allele was transferred to a CEN plasmid and expressed under the control of its own promoter. The defective transcription activation of this mutant was confirmed and extended to other Adr1-dependent genes by measuring RNA levels with quantitative real-time PCR (Figure 5). ADH2 expression was reduced 5-fold. Expression of the Adr1-dependent gene POX1 was reduced 2.9-fold. Two other Adr1-dependent genes, ATO3 and ADY2 were activated equally well by mutant and wild-type Adr1, consistent with the higher level of in vivo Adr1-binding at these promoters (Figure 4). Pho85 is involved in Adr1-Ser98 phosphorylation Binding and gene expression data showed that serine 98, proximal to the Zn-finger domains, is important for the function of Table 2. Beta-galactosidase assays on ADH2 promoter-lacZ with high-copy ADR1 wildtype or S98 mutants ß-galactosidase activity (Miller Units) Rb 340 (50)c 260 (47) 6 (0.1) DR 630 (170) 290 (100) 30 (3.0) straina Adr1 WT S98A S98D aTYY497 with pNKA1, pNKA2 or pNKA4 bR, repressed; DR, derepressed 0.05% glucose 2% ethanol, 2 hours cNumbers in parentheses are standard deviations for the average of three independent cultures doi:10.1371/journal.pone.0003213.t002 Figure 5. Activation of Adr1-dependent genes by Adr1 or the S98A mutant. RTqPCR on RNA isolated from Dadr1 strain CKY13 with pKD16-HA (wildtype, HA-tagged Adr1, black bars) and pKD16-HA-9 (S98A, HA-tagged Adr1, gray bars) derepressed in 0.05% glucose for 3 hours. Quantitation was for the indicated genes normalized to ACT1. Error bars are the standard deviation for the average of technical replicates. doi:10.1371/journal.pone.0003213.g005 Adr1. Since its phosphorylation state correlated with glucose availability, we identified potential kinases by screening for Adr1- phosphoserine 98 in a collection of 102 yeast strains, each deleted for a gene encoding a non-essential kinase. Each kinase mutant was transformed with a multi-copy plasmid expressing Adr1 from the ADH1 promoter. Phosphorylation of serine 98 was measured and normalized to total Adr1 using immunoprecipitation with anti-Adr1 antiserum followed by western blotting with either anti- pS98 or anti-Adr1 antiserum. Figure 6 shows the screening of a subset of mutants by western blot. For the wild-type strain, the signal ratio for the two antibodies was 0.17 (+/2 0.04, for three independent cultures). Three kinase mutations showed altered levels of serine 98 phosphorylation: pho85 (signal ratio 0.08), dun1 (0.27), and ssk2 (0.11). We focused on pho85 since it has been reported to have a role in growth control at the diauxic transition [24]. Dun1 is important for DNA damage control [25] and Ssk2 is involved in responding to osmotic stress [26]. Adr1 has not been implicated in either of these processes. Pho85 is a cyclin-dependent protein kinase with multiple cyclin partners [27]. Yeast strains with deletions in genes encoding Pho85-associated cyclins were taken from the yeast deletion collection and transformed with the multi-copy ADH1 promoter- ADR1 plasmid. Screening by ADHII activity assays [28] and Western blotting as described above, indicated that Pho80 might affect Adr1 phosphorylation and Adr1-dependent gene expression (data not shown). We then tested the effects of deleting the genes for the PHO85 kinase, its cyclin partner PHO80 and two cyclins Figure 6. Effect of Dpho85 on Adr1 phosphorylation. Western blots using antibody against phosphorylated serine 98 of Adr1 (a-pS98) or total Adr1 (a-Adr1) were performed on 99–125 mg of cell extract per lane from BY4741 wildtype or strains with the indicated kinase gene deletion. Each strain carried the high-copy Adr1 plasmid pNKA1-U. doi:10.1371/journal.pone.0003213.g006 PLoS ONE | www.plosone.org 5 September 2008 | Volume 3 | Issue 9 | e3213