Phosphorylation of Adr1 Figure 7. Model of the Adr1 DBD - DNA interaction, showing the position of serine 98. A model of the Adr1 PAR and DBD (ribbon structure) bound to its recognition sequence in DNA (space-filling model), based on NMR spectra of the complex in solution. Fingers 1 and 2 are the Zn-fingers; P87 and P97 are prolines; the arrow points to serine 98. Reprinted by permission from Macmillan Publishers Ltd: Nat Struct Biol 6:478–485, copyright 1999. doi:10.1371/journal.pone.0003213.g007 URA3 according to Cross [40]. pKD16-HA and pKD17-HA are pKD16 and pKD17 [5] tagged as described in Knop et al. [36]. The Serine 98 in pKD16-HA-9 was mutated to alanine using the QuickChange kit (Stratagene). In vitro phosphorylation of Adr1 Purification of Adr1 DBD (F1F2) or the S98A or S98D variants from E. coli was as described in Young et al. [4] from pF1F2, F1F2S98A or F1F2S98D. For in vitro phosphorylation of the Adr1 DBD, 1–10 mg of Adr1F1F2 was bound to Ni-NTA magnetic agarose beads (Qiagen) through its His6 tag, and resuspended in kinase buffer (10mM Tris pH 7.5, 10mM MgCl2, 20mM beta- mercaptoethanol or 10mM dithiothreitol, 0.005% Tween 20, 10mM imidazole, 2mM ATP, 1–5 mCi per reaction of 3000Ci/mmol [c-32P]-ATP). An equal volume of cell extract made according to Thukral et al. [30] was added, and after 15–60 minutes at 37uC, the reaction was stopped by washing the beads four times in 10mM Tris pH 8.0, 300mM NaCl, 10mM imidazole, 0.005% Tween 20. Adr1 DBD was eluted by boiling in 46 LDS loading buffer (Invitrogen), run on a 12% gel (Invitrogen) with protein standards, and visualized with a Storm Phosphorimager. Yeast protease inhibitor cocktail (Sigma), 1mM PMSF, and phosphatase inhibitors (1 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM ß-glycero- phosphate, 1 mM EGTA and 10 mM sodium fluoride), were added to extracts and wash buffers. Tryptic peptide mapping and phosphoamino acid analysis were performed as described in Boyle et al. [41] using Adr1 DBD phosphorylated as for the in vitro phosphorylation by cell extracts. The Pepsort program http://pingu.salk.edu/,sefton/Hyper_ protocols/pepsort1.html generated the graph with predicted tryptic peptide electrophoretic patterns. In vivo phosphorylation of Adr1 Gel electrophoresis and Western blots were carried out on yeast extracts generated according to Horvath and Reizman [42] and Hahn, S ‘‘Rapid yeast protein prep for SDS PAGE and Western,’’ http://www.fhcrc.org/science/labs/hahn/methods/biochem_meth/ yeast_prot_SDS.html with addition of a 30 second bead-beating step before boiling. Tris-Acetate 3–8% gels were run, Western blotted, visualized and quantitated according to manufacturer’s instructions for the NuPage system (Invitrogen) and Odyssey Infrared imaging system (Licor Biosciences). Polyclonal primary antibodies were used at 1:500–1:1000 and were anti-HA (Y-11, Santa Cruz Biochemicals), anti-Adr1 [10] or antibodies against phosphoserine 98 of Adr1. For the latter, the peptide LRLNGRTP(pS) GKLRSFVC and its unphosphorylated version were synthesized and used to generate and affinity purify the anti- pS98 antibody by Bethyl Laboratories (Montgomery, TX). Secondary IR-dye conjugated antibodies used at 1:1000–1:3000 were goat anti-mouse Alexa 680 (Molecular Probes) or IRdye800 conjugated anti-rabbit IgG (Rockland Immunochemicals). The screen for yeast mutants that affected Adr1 phosphorylation used a subset of 102 strains from the MATa yeast gene deletion collection (Invitrogen) that encode protein kinases according to the Saccharomyces Genome Database (yeastgenome.org). Strains were transformed with pNKA1-U. After growth under glucose repressing conditions, cell extracts were made by bead beating in lysis buffer (50mM Hepes pH 7.5, 140 mM NaCl, 1% Triton X-100, 0.1% PLoS ONE | www.plosone.org 7 September 2008 | Volume 3 | Issue 9 | e3213