Figure 4.
Figure 4. Overexpression of Swe1 blocks cells in metaphase. (A) Swe1 overexpression and latA induce similar levels of tyrosine phosphorylation on Cdk1. Wild-type (ADR4009) cells were treated with latA and GAL-SWE1 (ADR4289) cells were grown in YEP + 2% raffinose (Raff) and induced by addition of 2% galactose (Gal). Cells were harvested and immunoblotted for Swe1, Cdk1, and Cdk1-P-tyr. (B and C) Overexpression of Swe1 in mitosis stabilizes APC substrates and arrests cells with 2N DNA content. Wild-type (ADR2617), GAL-SWE1 (ADR3871), and GAL-SWE1 cdk1-Y19F (ADR4228) cells were grown in YEP + 2% raffinose, arrested with 10 µg/ml nocodazole, and induced with 2% galactose (t = 0) for 1 h. Nocodazole was washed out, and cells released into YEP + 2% galactose. 25 ng/ml α-factor was added (t = 150) to arrest cells in the following G1. Samples were taken for immunoblotting (A) and flow cytometry (B) at the indicated time points. (D) Swe1 inhibits Clb-associated kinase activity. Cells were grown as in A, and Cdk1–Clb2, –Clb3 or –Clb5 complexes were immunoprecipitated with anti-Clb antibodies and their histone H1 kinase activity was measured to assess Cdk1 inhibition. (E) Overexpression of Swe1 impairs sister chromatid separation. Wild-type (ADR1393) and GAL-SWE1 (ADR1395) strains were grown as in A, except 1 mM CuSO4 was added at t = −30 to induce expression of GFP-lacI, and the separation of sister lacO arrays at TRP1 (∼12 kb from CENIV) was visualized by fluorescence microscopy. The graphs in D and E are representative of one of three repeats. (F) Overexpressed Swe1 does not preferentially phosphorylate a particular Cdk1–cyclin complex. GAL-SWE1 cells (ADR3871) were grown as in A, and Cdk1–Clb2, –Clb3, or –Clb5 complexes were immunoprecipitated at the indicated time points and analyzed by immunoblotting with anti-Cdk1 and anti–Cdk1-P-tyr antibodies. (G) Overexpression of Swe1 in anaphase restabilizes APCCdc20 substrates. cdc15-2 (ADR4252) and cdc15-2 GAL-SWE1 (ADR4245) cells were grown in YEP + 2% raffinose and arrested in anaphase by temperature shift to 35°C, followed by induction with 2% galactose (t = 0). Parallel cultures were arrested in 10 µg/ml nocodazole (noc) at 25°C to illustrate peak levels of APC substrates. Cdk1 was used as a loading control for three independent membranes loaded with identical samples.

Overexpression of Swe1 blocks cells in metaphase. (A) Swe1 overexpression and latA induce similar levels of tyrosine phosphorylation on Cdk1. Wild-type (ADR4009) cells were treated with latA and GAL-SWE1 (ADR4289) cells were grown in YEP + 2% raffinose (Raff) and induced by addition of 2% galactose (Gal). Cells were harvested and immunoblotted for Swe1, Cdk1, and Cdk1-P-tyr. (B and C) Overexpression of Swe1 in mitosis stabilizes APC substrates and arrests cells with 2N DNA content. Wild-type (ADR2617), GAL-SWE1 (ADR3871), and GAL-SWE1 cdk1-Y19F (ADR4228) cells were grown in YEP + 2% raffinose, arrested with 10 µg/ml nocodazole, and induced with 2% galactose (t = 0) for 1 h. Nocodazole was washed out, and cells released into YEP + 2% galactose. 25 ng/ml α-factor was added (t = 150) to arrest cells in the following G1. Samples were taken for immunoblotting (A) and flow cytometry (B) at the indicated time points. (D) Swe1 inhibits Clb-associated kinase activity. Cells were grown as in A, and Cdk1–Clb2, –Clb3 or –Clb5 complexes were immunoprecipitated with anti-Clb antibodies and their histone H1 kinase activity was measured to assess Cdk1 inhibition. (E) Overexpression of Swe1 impairs sister chromatid separation. Wild-type (ADR1393) and GAL-SWE1 (ADR1395) strains were grown as in A, except 1 mM CuSO4 was added at t = −30 to induce expression of GFP-lacI, and the separation of sister lacO arrays at TRP1 (∼12 kb from CENIV) was visualized by fluorescence microscopy. The graphs in D and E are representative of one of three repeats. (F) Overexpressed Swe1 does not preferentially phosphorylate a particular Cdk1–cyclin complex. GAL-SWE1 cells (ADR3871) were grown as in A, and Cdk1–Clb2, –Clb3, or –Clb5 complexes were immunoprecipitated at the indicated time points and analyzed by immunoblotting with anti-Cdk1 and anti–Cdk1-P-tyr antibodies. (G) Overexpression of Swe1 in anaphase restabilizes APCCdc20 substrates. cdc15-2 (ADR4252) and cdc15-2 GAL-SWE1 (ADR4245) cells were grown in YEP + 2% raffinose and arrested in anaphase by temperature shift to 35°C, followed by induction with 2% galactose (t = 0). Parallel cultures were arrested in 10 µg/ml nocodazole (noc) at 25°C to illustrate peak levels of APC substrates. Cdk1 was used as a loading control for three independent membranes loaded with identical samples.

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