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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 4. Mps1-Mediated Phosphorylation of Hsp90 Is Essential for Mitotic Arrest.

Figure 4

(A) WT yHsp90-His6 yeast cells with endogenously Myc-epitope-tagged yMps1 were treated with 40 μM GB for indicated time points in liquid YPDA media. The stability of yMps1-Myc was assessed by immunoblotting.

(B) WT yHsp90-His6 yeast cells with endogenous yMps1-Myc were treated with either 40 μM GB for 24 hr or 50 mM proteasome inhibitor MG132 for 24 hr. Cells were also pretreated with MG132 1 hr prior to treatment with GB.

(C) Yeast cells expressing yHsp90-His6 (WT), T101A, and T101E with endogenous yMps1-Myc were treated with 100 μg/ml CHX. yHsp90-His6 and yMps1-Myc proteins were detected by immunoblotting at the indicated times. Data were also quantified and presented as bar charts. Error bars represent SD. OD, optical density.

(D) Yeast with yHsp90-His6 (WT), T101A-yHsp90-His6 (T101A), and T101E-yHsp90-His6 (T101E) mutants with endogenous yMps1-Myc were used to isolate yHsp90-His6 by Ni-NTA, and interaction of yMps1 was examined by immunoblotting. Empty plasmid was used as a control (C).

(E) HEK293 cells were transfected with empty plasmid (C), hHsp90α-FLAG (WT), T115A-hHsp90α-FLAG (T115A), or T115E-hHsp90α-FLAG (T115E). Hsp90-FLAG was immunoprecipitated (IP) with anti-FLAG M2 affinity gel, and interaction of human Mps1 was examined by immunoblotting.

(F) yMps1-Myc was isolated from yeast cells expressing yHsp90-His6 (WT), T101A-yHsp90-His6 (T101A), and T101E-yHsp90-His6 (T101E). yMps1 activity was examined by immunoblotting using pan-phospho-threonine antibody.

(G) Flow cytometry analysis of yHsp90-His6 (WT), T101A-yHsp90-His6 (T101A), and T101E-yHsp90-His6 (T101E) mutants grown in liquid YPDA over-night at 25°C. 57% of WT, 54% of T101A, and 77% of T101E cells were arrested in G2.

(H) yHsp90-His6 (WT) and T101A-yHsp90-His6 (T101A) cells containing GAL-yMPS1-GST were grown in liquid YP-raffinose overnight at 25°C and then transferred to YP-galactose for 2 hr at 25°C. Flow cytometry analysis revealed only WT cell accumulation in G2 (75%).

(I) Budding morphology of distributions in WT, T101, and mps1-1 yeast mutants. Cells were arrested in G1 with an α-factor at 25°C and then released in medium containing 20 μg/ml nocodazole for 4 hr at either 25°C or 37°C. 100 cells were scored for large buds (L), multiply budded (M), and unbudded (U). Cells were scored from three independent experiments.

All data represent mean ± SD. **p < 0.005.