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The Bub2-dependent mitotic pathway in yeast acts every cell cycle and regulates cytokinesis

¹ Division of Yeast Genetics, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA, UK
² Cyclacel/Polgen, Babraham Hall, Babraham, Cambridgeshire, CB2 4AT, UK
³ Centre de Recherche de Biochimie Macromoleculaire, CNRS, UPR 1086, 1919, Route de Mende, 34293 Montpellier Cedex 5, France

View larger version (24K): [in a new window]   Fig. 1. Bfa1 and Bub2 physically associate. (A) Bfa1 negatively regulates Dbf2 kinase activation. Isogenic strains carrying integrated DBF2-6MYC, YDF20 (apc2-8) and SLY103 (apc2-8 bfa1) were grown to mid-log phase at 25°C and arrested with -factor (F) or with nocodazole at 37°C (Noco at 37°C) or at 37°C (37°C) for 3 hours. Extracts were immunoprecipitated with anti-Myc (9E10) and Dbf2 kinase assayed (lower panel). The upper part of the kinase assay gel was immunoblotted and probed with 9E10 antibody to control for loading. (B) Bfa1 and Bub2 associate across the cell cycle. Strain SLY106 (BUB2-13MYC 3HA-BFA1) was grown to mid-log and synchronised with -factor. Cells were sampled for protein extraction at the times shown. 300 µg extract was immunoprecipitated using anti-HA (12CA5) antibody, analysed using SDS-PAGE, immunoblotted and probed with 9E10. Lanes 1-3 show an untagged control (CG378) and two singly tagged strains. The Ig band serves as a loading control. The bottom panel shows budding in the synchronised culture.

View larger version (41K): [in a new window]   Fig. 2. Bfa1 and Bub2 physically associate with Tem1. pGAL-6HIS-TEM1 was introduced into strain SLY106 (BUB2-13MYC 3HA-BFA1) and control strain CG378. Mid-log cells (ML) grown with or without galactose (lanes 1-4) were sampled. The culture of strain SLY106 with galactose was divided and -factor (F), HU or nocodazole (Noco) added and incubation continued for 3 hours before sampling. Crude extracts were prepared and incubated with nickel beads. Proteins binding to the beads were eluted and an immunoblot prepared. This was probed with the antibodies shown. Note that strain CG378 is congenic, not isogenic, with strain SLY106, which may account for the high level of Tem1 in lane 1.

View larger version (17K): [in a new window]   Fig. 3. Bfa1 is a phosphoprotein. (A) Mid-log cells of strain SLY105 (3HA-BFA1) were harvested or arrested by using -factor (F), HU or nocodazole (Noco) for 3 hours before harvesting. Following protein extraction, an immunoblot was prepared and probed with 12CA5. The Roman numerals on the right indicate the various Bfa1 species. (B) Mid-log cells of strain SLY105 were harvested immediately or following treatment with nocodazole for 3 hours. Protein extraction was performed as usual or in lysis buffer without phosphatase inhibitors (+/- PI). 70 µg extract was incubated with calf alkaline phosphatase (CAP) at 30°C for 30 minutes prior to SDS-PAGE and immunoblotting. (C) Phosphorylation of Bfa1 is dependent upon Bub2. Strain SLY111, isogenic to SLY105 but containing bub2, was treated as described in (A).

View larger version (26K): [in a new window]   Fig. 4. Bfa1 undergoes cell-cycle-dependent phosphorylation that is enhanced following nocodazole treatment. Strain SLY105 (3HA-BFA1) was synchronised with -factor and released into fresh medium in the absence (A) or presence (B) of nocodazole. Samples for protein extraction were taken at the indicated times after release. Extracts were immunoblotted and probed with anti-HA or anti-Clb2 as indicated. The right panel shows budding curves for the two cultures.

View larger version (26K): [in a new window]   Fig. 5. Lte1 undergoes cell-cycle-dependent phosphorylation that is maintained following nocodazole treatment. Strain SJY121 (LTE1-3HA) was synchronised with -factor and released into fresh medium at 25°C in the absence (A) or presence (B) of nocodazole. Samples for protein extraction were taken at the indicated times after release. Immunoblots were analysed with anti-HA or anti-Clb2 as indicated. The lower panel shows budding curves for the two cultures.

View larger version (34K): [in a new window]   Fig. 6. Cdc5 controls Bfa1 and Lte1 phosphorylation. (A) Strain SLY109 (3HA-BFA1 cdc5ts (msd2-1)) was grown to mid-log phase at 25 °C and sampled. Some of the remaining cells were transferred to 37°C and nocodazole was added to others with incubation at either 25°C or 37°C. Following protein extraction an immunoblot was prepared and probed with 12CA5. (B) Strain SLY109 was synchronised with -factor and released at either 25°C or 37°C. Samples were removed at the times indicated and an immunoblot prepared. The lower panel shows budding curves for the two cultures. (C) Mid-log cells of strain SLY109 were treated with nocodazole for 3 hours at 25°C. Incubation of one half of the culture was continued at this temperature, whereas the other half was transferred to 37°C. After sampling, an immunoblot was prepared from the cells. (D) Strains SJY121 and 122 were grown to mid-log phase at 25°C. The cultures were split and one half was incubated with nocodazole. Following 3 hours incubation at 30°C, cells were harvested and an immunoblot prepared from the extract. Note that longer exposure did not reveal further phosphorylations in lanes 1 and 2.

View larger version (52K): [in a new window]   Fig. 7. Bub2/Bfa1 act during metaphase arrest caused by means other than spindle damage. (A) Bfa1 shows the same pattern of phosphorylation during apc2-8 temperature arrest as after nocodazole-induced arrest. Three strains expressing 3HA-Bfa1, a wild type, SLY116 (apc2-8) and JB5A1-51C (apc2-8 cdc5) were grown to mid-log at 25°C and transferred to 37°C for 3 hours in the presence or absence of nocodazole and an immunoblot prepared. (B) Bub2 restrains actin ring formation in metaphase arrest caused by apc2-8 temperature shift or overexpression of indestructible Pds1. Mid-log cultures of the apc2-8 strains were transferred to 37°C for 3 hours. For overexpression of indesructible Pds1, the strains were grown to mid-log phase in YEP sucrose, arrested with -factor and released into fresh YEP sucrose and galactose added to 2% when all cells had budded. Once large-budded cells had accumulated, samples were removed for microscopy. Cells were stained with DAPI and rhodamine-phalloidin for actin distribution (Frenz et al., 2000). The cells shown above (B) are representative examples of the large-budded cells examined. (C, D) Bub2 restrains mitotic exit in apc2-8-arrested cells. Strains containing TUB1.GFP in an apc2-8 or apc2-8 bub2 background were grown to mid-log phase at 23°C, shifted to 37°C and sampled at 2 hour intervals. Rebudding was assessed microscopically following sonication (C) and the state of the spindles and SPBs examined by fluorescent microscopy (D).

View larger version (11K): [in a new window]   Fig. 8. Metaphase arrest activates Bfa1/Bub2 to inhibit Tem1 and shut down the MEN. (A) Metaphase arrest induced by SAC activation or the apc2-8 mutation leads to a presumed activating phosphorylation of Bfa1. This Bfa1 phosphorylation, together with phosphorylation of Lte1, inactivate Tem1 to block activation of the MEN (including Dbf2) and cytokinesis (Frenz et al., 2000). (B) Bub2/Bfa1 control of Tem1 is rate limiting in metaphase to block MEN activation. Lte1 positive control of Tem1 occurs in anaphase and promotes mitotic exit.