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Orchestrating anaphase and mitotic exit: separase cleavage and localization of Slk19

Nature Cell Biology volume 3, pages 771–777 (2001)Cite this article

Abstract

Anaphase in budding yeast is triggered by cleavage of the central subunit, Scc1, of the chromosomal cohesin complex by the protease separase. Here we show that separase also cleaves the kinetochore-associated protein Slk19 at anaphase onset. Separase activity is also required for the proper localization of a stable Slk19 cleavage product to the spindle midzone in anaphase. The cleavage and localization of Slk19 are necessary to stabilize the anaphase spindle, and we show that a stable spindle is a prerequisite for timely exit from mitosis. This demonstrates the cleavage of targets other than cohesin by separase in the orchestration of high-fidelity anaphase.

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Figure 1: Slk19 is a cleavage target of separase.
Figure 2: Phenotype of uncleavable Slk19.
Figure 3: The Slk19 cleavage product is required for spindle stability in anaphase.
Figure 4: Delay of mitotic exit after spindle breakdown.
Figure 5: Cleavage of Slk19 by TEV protease.
Figure 6: Separase activity in anaphase is required for Slk19 and Ase1 localization.

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References

  1. Nasmyth, K., Peters, J.-M. & Uhlmann, F. Splitting the chromosome: cutting the ties that bind sister chromatids. Science 288, 1379–1384 (2000).

    Article  CAS  Google Scholar 

  2. Hirano, T. Chromosome cohesion, condensation, and separation. Annu. Rev. Biochem. 69, 115–144 (2000).

    Article  CAS  Google Scholar 

  3. Koshland, D. E. & Guacci, V. Sister chromatid cohesion: the beginning of a long and beautiful relationship. Curr. Opin. Cell Biol. 12, 297–301 (2000).

    Article  CAS  Google Scholar 

  4. Uhlmann, F., Lottspeich, F. & Nasmyth, K. Sister-chromatid separation at anaphase onset is promoted by cleavage of the cohesin subunit Scc1. Nature 400, 37–42 (1999).

    Article  CAS  Google Scholar 

  5. Uhlmann, F., Wernic, D., Poupart, M.-A., Koonin, E. V. & Nasmyth, K. Cleavage of cohesin by the CD clan protease separin triggers anaphase in yeast. Cell 103, 375–386 (2000).

    Article  CAS  Google Scholar 

  6. Uhlmann, F. Secured cutting: controlling separase at the metaphase to anaphase transition. EMBO Rep. 2, 487–492 (2001).

    Article  CAS  Google Scholar 

  7. Bachmair, A., Finley, D. & Varshavsky, A. In vivo half-life of a protein is a function of its amino-terminal residue. Science 234, 179–186 (1986).

    Article  CAS  Google Scholar 

  8. Varshavsky, A. The N-end rule: functions, mysteries, uses. Proc. Natl Acad. Sci. USA 93, 12142–12149 (1996).

    Article  CAS  Google Scholar 

  9. Rao, H., Uhlmann, F., Nasmyth, K. & Varshavsky, A. Degradation of a cohesin subunit by the N-end rule pathway is essential for chromosome stability. Nature 410, 955–959 (2001).

    Article  CAS  Google Scholar 

  10. Winey, M. et al. Three-dimensional ultrastructural analysis of the Saccharomyces cerevisiae mitotic spindle. J. Cell Biol. 129, 1601–1615 (1995).

    Article  CAS  Google Scholar 

  11. Winey, M. & O'Toole, E. T. The spindle cycle in budding yeast. Nature Cell Biol. 3, E23–E27 (2001).

    Article  CAS  Google Scholar 

  12. Bardin, A. J., Visintin, R. & Amon, A. A mechanism for coupling exit from mitosis to partitioning of the nucleus. Cell 102, 21–31 (2000).

    Article  CAS  Google Scholar 

  13. Pereira, G., Höfken, T., Grindlay, J., Manson, C. & Schiebel, E. The Bub2p spindle checkpoint links nuclear migration with mitotic exit. Mol. Cell 6, 1–10 (2000).

    Article  CAS  Google Scholar 

  14. McCollum, D. & Gould, K. L. Timing is everything: regulation of mitotic exit and cytokinesis by the MEN and SIN. Trends Cell Biol. 11, 89–95 (2001).

    Article  CAS  Google Scholar 

  15. Funabiki, H., Kumada, K. & Yanagida, M. Fission yeast Cut1 and Cut2 are essential for sister chromatid separation, concentrate along the metaphase spindle and form large complexes. EMBO J. 15, 6617–6628 (1996).

    Article  CAS  Google Scholar 

  16. Ciosk, R. et al. An Esp1/Pds1 complex regulates loss of sister chromatid cohesion at the metaphase to anaphase transition in yeast. Cell 93, 1067–1076 (1998).

    Article  CAS  Google Scholar 

  17. Kumada, K. et al. Cut1 is loaded onto the spindle by binding to Cut2 and promotes anaphase spindle movement upon Cut2 proteolysis. Curr. Biol. 8, 633–641 (1998).

    Article  CAS  Google Scholar 

  18. Jensen, S., Segal, M., Clarke, D. J. & Reed, S. I. A novel role of the budding yeast separin Esp1 in anaphase spindle elongation: evidence that proper spindle association of Esp1 is regulated by Pds1. J. Cell Biol. 152, 27–40 (2001).

    Article  CAS  Google Scholar 

  19. Zeng, X. et al. Slk19p is a centromere protein that functions to stabilize mitotic spindles. J. Cell Biol. 146, 415–425 (1999).

    Article  CAS  Google Scholar 

  20. Buonomo, S. B. C. et al. Disjunction of homologous chromosomes in meiosis I depends on proteolytic cleavage of the meiotic cohesin Rec8 by separin. Cell 103, 387–398 (2000).

    Article  CAS  Google Scholar 

  21. Alexandru, G., Uhlmann, F., Poupart, M.-A., Mechtler, K. & Nasmyth, K. Phosphorylation of the cohesin subunit Scc1 by Polo/Cdc5 kinase regulates sister chromatid separation in yeast. Cell 105, 459–472 (2001).

    Article  CAS  Google Scholar 

  22. Hieter, P., Mann, C., Snyder, M. & Davis, R. W. Mitotic stability of yeast chromosomes: a colony color assay that measures nondisjunction and chromosome loss. Cell 40, 381–392 (1985).

    Article  CAS  Google Scholar 

  23. Fitch, I. T. et al. Characterization of four B-type cyclin genes of the budding yeast Saccharomyces cerevisiae. Mol. Biol. Cell 3, 805–818 (1992).

    Article  CAS  Google Scholar 

  24. Juang, Y.-L. et al. APC-mediated proteolysis of Ase1 and the morphogenesis of the mitotic spindle. Science 275, 1311–1314 (1997).

    Article  CAS  Google Scholar 

  25. Tinker-Kulberg, R. L. & Morgan, D. O. Pds1 and Esp1 control both anaphase and mitotic exit in normal cells and after DNA damage. Genes Dev. 13, 1936–1949 (1999).

    Article  CAS  Google Scholar 

  26. Tomonaga, T. et al. Characterization of fission yeast cohesin: essential anaphase proteolysis of Rad21 phosphorylated in the S phase. Genes Dev. 14, 2757–2770 (2000).

    Article  CAS  Google Scholar 

  27. Waizenegger, I. C., Hauf, S., Meinke, A. & Peters, J.-M. Two distinct pathways remove mammalian cohesin complexes from chromosome arms in prophase and from centromeres in anaphase. Cell 103, 399–410 (2000).

    Article  CAS  Google Scholar 

  28. Funabiki, H. & Murray, A. W. The Xenopus chromokinesin Xkid is essential for metaphase chromosome alignment and must be degraded to allow anaphase chromosome movement. Cell 102, 411–424 (2000).

    Article  CAS  Google Scholar 

  29. Abrieu, A., Kahana, J. A., Wood, K. W. & Cleveland, D. W. CENP-E as an essential component of the mitotic checkpoint in vitro. Cell 102, 817–826 (2000).

    Article  CAS  Google Scholar 

  30. Gietz, R. D. & Sugino, A. New yeast–Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base restriction sites. Gene 74, 527–534 (1988).

    Article  CAS  Google Scholar 

  31. Dougherty, W. G., Cary, S. M. & Parks, T. D. Molecular genetic analysis of a plant virus polyprotein cleavage site: a model. Virology 171, 356–364 (1989).

    Article  CAS  Google Scholar 

  32. Knop, M. et al. Epitope tagging of yeast genes using a PCR-based strategy: more tags and improved practical routines. Yeast 15, 963–972 (1999).

    Article  CAS  Google Scholar 

  33. Wach, A., Brachat, A., Pöhlmann, R. & Philippsen, P. New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 10, 1793–1808 (1994).

    Article  CAS  Google Scholar 

  34. Rose, M. D., Winston, F. & Hieter, P. Laboratory Course Manual for Methods in Yeast Genetics (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1990).

    Google Scholar 

  35. Spellman, P. T. et al. Comprehensive identification of cell cycle-regulated genes of the yeast Saccharomyces cerevisiae by microarray hybridization. Mol. Biol. Cell 9, 3273–3297 (1998).

    Article  CAS  Google Scholar 

  36. Hagan, I. M. & Ayscough, K. R. in Protein Localization by Fluorescence Microscopy (ed. Allan, V. J.) 179–208 (Oxford Univ. Press, 2000).

    Google Scholar 

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Acknowledgements

We thank J. Kilmartin for his gift of the anti-Tub4 antibody, K. Sawin for the anti-GFP antibody, K. Nasmyth for yeast strains and for his support and encouragement, and J. Diffley and T. Toda for critical comments on the manuscript.

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  1. Chromosome Segregation Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London, WC2A 3PX, UK

    Matthew Sullivan, Christine Lehane & Frank Uhlmann

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Correspondence to Frank Uhlmann.

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Sullivan, M., Lehane, C. & Uhlmann, F. Orchestrating anaphase and mitotic exit: separase cleavage and localization of Slk19. Nat Cell Biol 3, 771–777 (2001). https://doi.org/10.1038/ncb0901-771

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