Fig. 5

Sch9 phosphorylates Hsf1 in glucose-rich conditions. a–c Hsf1 phosphorylation is affected by the absence of Ssb or functional Sch9. Strains as indicated were grown on glucose (+glucose) or were glucose depleted for 10 min (−glucose) prior to collection. Protein extracts were analyzed via phos-tag gels followed by immunoblotting with α-Hsf1. H1/H2: predominant Hsf1 species in glucose-grown wild-type (a, b) and Δyak1 (c) cells; H1*/H2*: faster migrating species in Δssb1Δssb2 cells, Δsch9 cells, or in cells expressing non-functional Sch9 versions as indicated. H3: predominant hyper-phosphorylated Hsf1 species formed upon glucose depletion. H3*: hypo-phosphorylated Sch9 species form in cells lacking Ssb or active Sch9 upon glucose depletion. Arrows indicate overexpression of Sch9 or Yak1, respectively. d Cartoon summary of Hsf1 phosphorylation in glucose-rich and glucose-starved conditions. In glucose-grown wild-type cells Hsf1 is phosphorylated to H1/H2. In glucose-grown Δssb1Δssb2 cells, or in cells lacking functional Sch9, Hsf1 is hypo-phosphorylated to H1*/H2*. Yak1, activated upon glucose depletion, efficiently phosphorylates H1/H2 to H3, however, does not efficiently phosphorylate the hypo-phosphorylated species H1*/H2*. e Sch9 phosphorylates Hsf1 in vitro. A mixture of partially purified His₆-sumo-Hsf1 and GST-Maf1 was incubated with Sch9-2D3E-FLAG or Sch9-5A-FLAG in the presence of γ-³²P-ATP (see Methods section). The total amount Sch9-2D3E-FLAG or Sch9-5A-FLAG in the reaction was analyzed via immunoblotting with α-FLAG (upper panel), total proteins in the reactions were visualized by Coomassie staining (middle panel, and see also Supplementary Fig. 5), incorporation of ³²P was analyzed via exposure of the Coomassie stained gel to a phosphor image plate (lower panel)