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. Author manuscript; available in PMC: 2016 May 31.
Published in final edited form as: Cell Rep. 2016 Jan 21;14(4):872–884. doi: 10.1016/j.celrep.2015.12.084

Figure 1. Phosphorylation of a Conserved Threonine Residue in the N-Domain of Yeast and Human Hsp90.

Figure 1

(A) Pymol cartoon of the Hsp90 monomer without Sba1 (PDB: 2CG9). The cyan region represents the N-terminal domain (NTD), yellow represents the middle domain (MD), and orange represents the C-terminal domain (CTD). The PreScission protease region is highlighted in pink. Alignment of yeast and human Hsp90 showing the conservation of T101 and T115 amino acid residues.

(B and C) N-domain phosphorylation of (B) WT yHsp90-His6 (WT) and T101A-yHsp90-His6 (T101A) or (C) WT hHsp90α-FLAG (WT) and T115A-hHsp90α-FLAG (T115A) detected by immunoblotting with anti-hexahistidine for yHsp90 and anti-FLAG for hHsp90α and pan-anti-phosphothreonine antibodies after PreScission protease digestion. IP, immunoprecipitation; Phos-Thr, phospho-threonine.

(D) Yeast cells expressing WT yHsp90-His6 (WT) or T101A-yHsp90-His6 (T101A) and carrying yMPS1-MYC under GAL1-promoter were grown on raffinose (−) or galactose (+). N-domain threonine phosphorylation was assessed after pull-downs and after PreScission protease digestion and immunoblotting.

(E) yMps1 mediates threonine phosphorylation of WT yHsp90 and T101A-yHsp90 (T101A) mutant in vitro. Bacterially expressed and purified yHsp90-His6 and T101A mutant were used as substrates for yMps1. Threonine phosphorylation was detected by immunoblotting with pan-anti-phospho-threonine antibody. SE, short exposure; LE, long exposure.

(F) Bacterially expressed and purified hHsp90α-His6 and T115A mutant were used as substrates for hMps1. Threonine phosphorylation was detected by immunoblotting with pan-anti-phospho-threonine antibody.

See also Figures S1 and S2.