Fig. 3

Sch9 kinase activity is disturbed in cells lacking Ssb. a, b FLAG-IP reactions were performed with extracts derived from Δsch9 cells overexpressing either Sch9 (−FLAG) or Sch9-FLAG (+FLAG). If indicated ATP was added at a concentration of 10 mM. Total extracts (input) and immunoprecipitated proteins (FLAG-IP) were separated on Tris-Tricine gels and were subsequently analyzed via immunoblotting using α-Sch9, α-Ssb, and α-Maf1. Details are described in Methods section and in Supplementary Fig. 3a, b. c, d Maf1 phosphorylation is reduced in Δssb1Δssb2 cells. Strains as indicated were grown on glucose (+glucose) or were depleted for glucose for 10 min (−glucose) prior to collection. Strains labeled with Sch9↑ overexpress Sch9. Protein extracts were analyzed via phos-tag gels followed by immunoblotting with α-Maf1. M0: dephosphorylated Maf1 species, M1: hyper-phosphorylated Maf1 species. λP: lambda phosphatase treated extract. e Protein levels of Sch9 and Maf1 are unaffected in cells lacking Ssb. Extracts from glucose-grown strains were resolved on a Tris-Tricine gel and were subsequently analyzed via immunoblotting with α-Maf1, α-Sse1, α-Ssb, or α-Sch9. The numbers below the Maf1 bands indicate the intensity of the total Maf1 signal in Δssb1Δssb2 cells relative to that in wild-type cells. f Maf1 hyper-phosphorylation requires phosphorylation of Sch9-T737 and the Ssb co-chaperone RAC. Protein extracts from indicated strain were analyzed via phos-tag gels followed by immunoblotting with α-Maf1. g Maf1 phosphorylation requires ATP-hydrolysis by Ssb. Analysis as in f. h Overexpression of Ssa does not restore Maf1 hyper-phosphorylation in cells lacking Ssb. Aliquots of total cell extracts as indicated were separated via phos-tag gels (upper panel) or Tris-Tricine gels (lower panel) and were analyzed via immunoblotting with α-Maf1, α-Ssa, or α-Sse1 (loading control). i Cartoon summary of Maf1 phosphorylation in wild-type and Δssb1Δssb2 cells. In glucose-rich conditions Maf1 is hyper-phosphorylated by Sch9-S3, while Sch9-S2 and Sch9-S4, formed upon glucose depletion are inactive towards Maf1. Sch9-S3^# in Δssb1Δssb2 cells, lacking phosphorylation at T737, is not fully active with respect to Maf1 phosphorylation. For more details compare Results section