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. 2001 Apr;157(4):1493–1502. doi: 10.1093/genetics/157.4.1493

The spindle checkpoint of the yeast Saccharomyces cerevisiae requires kinetochore function and maps to the CBF3 domain.

R D Gardner 1, A Poddar 1, C Yellman 1, P A Tavormina 1, M C Monteagudo 1, D J Burke 1
PMCID: PMC1461604  PMID: 11290706

Abstract

We have measured the activity of the spindle checkpoint in null mutants lacking kinetochore activity in the yeast Saccharomyces cerevisiae. We constructed deletion mutants for nonessential genes by one-step gene replacements. We constructed heterozygous deletions of one copy of essential genes in diploid cells and purified spores containing the deletion allele. In addition, we made gene fusions for three essential genes to target the encoded proteins for proteolysis (degron alleles). We determined that Ndc10p, Ctf13p, and Cep3p are required for checkpoint activity. In contrast, cells lacking Cbf1p, Ctf19p, Mcm21p, Slk19p, Cse4p, Mif2p, Mck1p, and Kar3p are checkpoint proficient. We conclude that the kinetochore plays a critical role in checkpoint signaling in S. cerevisiae. Spindle checkpoint activity maps to a discreet domain within the kinetochore and depends on the CBF3 protein complex.

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Selected References

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  1. Adams A. E., Pringle J. R. Relationship of actin and tubulin distribution to bud growth in wild-type and morphogenetic-mutant Saccharomyces cerevisiae. J Cell Biol. 1984 Mar;98(3):934–945. doi: 10.1083/jcb.98.3.934. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Amon A. The spindle checkpoint. Curr Opin Genet Dev. 1999 Feb;9(1):69–75. doi: 10.1016/s0959-437x(99)80010-0. [DOI] [PubMed] [Google Scholar]
  3. Bai C., Sen P., Hofmann K., Ma L., Goebl M., Harper J. W., Elledge S. J. SKP1 connects cell cycle regulators to the ubiquitin proteolysis machinery through a novel motif, the F-box. Cell. 1996 Jul 26;86(2):263–274. doi: 10.1016/s0092-8674(00)80098-7. [DOI] [PubMed] [Google Scholar]
  4. Basu J., Bousbaa H., Logarinho E., Li Z., Williams B. C., Lopes C., Sunkel C. E., Goldberg M. L. Mutations in the essential spindle checkpoint gene bub1 cause chromosome missegregation and fail to block apoptosis in Drosophila. J Cell Biol. 1999 Jul 12;146(1):13–28. doi: 10.1083/jcb.146.1.13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bernard P., Hardwick K., Javerzat J. P. Fission yeast bub1 is a mitotic centromere protein essential for the spindle checkpoint and the preservation of correct ploidy through mitosis. J Cell Biol. 1998 Dec 28;143(7):1775–1787. doi: 10.1083/jcb.143.7.1775. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brown M. T., Goetsch L., Hartwell L. H. MIF2 is required for mitotic spindle integrity during anaphase spindle elongation in Saccharomyces cerevisiae. J Cell Biol. 1993 Oct;123(2):387–403. doi: 10.1083/jcb.123.2.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Burke D. J. Complexity in the spindle checkpoint. Curr Opin Genet Dev. 2000 Feb;10(1):26–31. doi: 10.1016/s0959-437x(99)00040-4. [DOI] [PubMed] [Google Scholar]
  8. Cahill D. P., Lengauer C., Yu J., Riggins G. J., Willson J. K., Markowitz S. D., Kinzler K. W., Vogelstein B. Mutations of mitotic checkpoint genes in human cancers. Nature. 1998 Mar 19;392(6673):300–303. doi: 10.1038/32688. [DOI] [PubMed] [Google Scholar]
  9. Chan G. K., Jablonski S. A., Sudakin V., Hittle J. C., Yen T. J. Human BUBR1 is a mitotic checkpoint kinase that monitors CENP-E functions at kinetochores and binds the cyclosome/APC. J Cell Biol. 1999 Sep 6;146(5):941–954. doi: 10.1083/jcb.146.5.941. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Connelly C., Hieter P. Budding yeast SKP1 encodes an evolutionarily conserved kinetochore protein required for cell cycle progression. Cell. 1996 Jul 26;86(2):275–285. doi: 10.1016/S0092-8674(00)80099-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Doheny K. F., Sorger P. K., Hyman A. A., Tugendreich S., Spencer F., Hieter P. Identification of essential components of the S. cerevisiae kinetochore. Cell. 1993 May 21;73(4):761–774. doi: 10.1016/0092-8674(93)90255-O. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dohmen R. J., Wu P., Varshavsky A. Heat-inducible degron: a method for constructing temperature-sensitive mutants. Science. 1994 Mar 4;263(5151):1273–1276. doi: 10.1126/science.8122109. [DOI] [PubMed] [Google Scholar]
  13. Gardner R. D., Burke D. J. The spindle checkpoint: two transitions, two pathways. Trends Cell Biol. 2000 Apr;10(4):154–158. doi: 10.1016/s0962-8924(00)01727-x. [DOI] [PubMed] [Google Scholar]
  14. Gorbsky G. J., Chen R. H., Murray A. W. Microinjection of antibody to Mad2 protein into mammalian cells in mitosis induces premature anaphase. J Cell Biol. 1998 Jun 1;141(5):1193–1205. doi: 10.1083/jcb.141.5.1193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hardwick K. G., Li R., Mistrot C., Chen R. H., Dann P., Rudner A., Murray A. W. Lesions in many different spindle components activate the spindle checkpoint in the budding yeast Saccharomyces cerevisiae. Genetics. 1999 Jun;152(2):509–518. doi: 10.1093/genetics/152.2.509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Herman P. K., Rine J. Yeast spore germination: a requirement for Ras protein activity during re-entry into the cell cycle. EMBO J. 1997 Oct 15;16(20):6171–6181. doi: 10.1093/emboj/16.20.6171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hyland K. M., Kingsbury J., Koshland D., Hieter P. Ctf19p: A novel kinetochore protein in Saccharomyces cerevisiae and a potential link between the kinetochore and mitotic spindle. J Cell Biol. 1999 Apr 5;145(1):15–28. doi: 10.1083/jcb.145.1.15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jiang W., Lim M. Y., Yoon H. J., Thorner J., Martin G. S., Carbon J. Overexpression of the yeast MCK1 protein kinase suppresses conditional mutations in centromere-binding protein genes CBF2 and CBF5. Mol Gen Genet. 1995 Feb 6;246(3):360–366. doi: 10.1007/BF00288609. [DOI] [PubMed] [Google Scholar]
  19. Kallio M., Weinstein J., Daum J. R., Burke D. J., Gorbsky G. J. Mammalian p55CDC mediates association of the spindle checkpoint protein Mad2 with the cyclosome/anaphase-promoting complex, and is involved in regulating anaphase onset and late mitotic events. J Cell Biol. 1998 Jun 15;141(6):1393–1406. doi: 10.1083/jcb.141.6.1393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Labib K., Diffley J. F., Kearsey S. E. G1-phase and B-type cyclins exclude the DNA-replication factor Mcm4 from the nucleus. Nat Cell Biol. 1999 Nov;1(7):415–422. doi: 10.1038/15649. [DOI] [PubMed] [Google Scholar]
  21. Meluh P. B., Yang P., Glowczewski L., Koshland D., Smith M. M. Cse4p is a component of the core centromere of Saccharomyces cerevisiae. Cell. 1998 Sep 4;94(5):607–613. doi: 10.1016/s0092-8674(00)81602-5. [DOI] [PubMed] [Google Scholar]
  22. Middleton K., Carbon J. KAR3-encoded kinesin is a minus-end-directed motor that functions with centromere binding proteins (CBF3) on an in vitro yeast kinetochore. Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):7212–7216. doi: 10.1073/pnas.91.15.7212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ortiz J., Stemmann O., Rank S., Lechner J. A putative protein complex consisting of Ctf19, Mcm21, and Okp1 represents a missing link in the budding yeast kinetochore. Genes Dev. 1999 May 1;13(9):1140–1155. doi: 10.1101/gad.13.9.1140. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Poddar A., Roy N., Sinha P. MCM21 and MCM22, two novel genes of the yeast Saccharomyces cerevisiae are required for chromosome transmission. Mol Microbiol. 1999 Jan;31(1):349–360. doi: 10.1046/j.1365-2958.1999.01179.x. [DOI] [PubMed] [Google Scholar]
  25. Russell I. D., Grancell A. S., Sorger P. K. The unstable F-box protein p58-Ctf13 forms the structural core of the CBF3 kinetochore complex. J Cell Biol. 1999 May 31;145(5):933–950. doi: 10.1083/jcb.145.5.933. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sassoon I., Severin F. F., Andrews P. D., Taba M. R., Kaplan K. B., Ashford A. J., Stark M. J., Sorger P. K., Hyman A. A. Regulation of Saccharomyces cerevisiae kinetochores by the type 1 phosphatase Glc7p. Genes Dev. 1999 Mar 1;13(5):545–555. doi: 10.1101/gad.13.5.545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Skibbens R. V., Hieter P. Kinetochores and the checkpoint mechanism that monitors for defects in the chromosome segregation machinery. Annu Rev Genet. 1998;32:307–337. doi: 10.1146/annurev.genet.32.1.307. [DOI] [PubMed] [Google Scholar]
  28. Stoler S., Keith K. C., Curnick K. E., Fitzgerald-Hayes M. A mutation in CSE4, an essential gene encoding a novel chromatin-associated protein in yeast, causes chromosome nondisjunction and cell cycle arrest at mitosis. Genes Dev. 1995 Mar 1;9(5):573–586. doi: 10.1101/gad.9.5.573. [DOI] [PubMed] [Google Scholar]
  29. Strunnikov A. V., Kingsbury J., Koshland D. CEP3 encodes a centromere protein of Saccharomyces cerevisiae. J Cell Biol. 1995 Mar;128(5):749–760. doi: 10.1083/jcb.128.5.749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Tavormina P. A., Burke D. J. Cell cycle arrest in cdc20 mutants of Saccharomyces cerevisiae is independent of Ndc10p and kinetochore function but requires a subset of spindle checkpoint genes. Genetics. 1998 Apr;148(4):1701–1713. doi: 10.1093/genetics/148.4.1701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Tavormina P. A., Wang Y., Burke D. J. Differential requirements for DNA replication in the activation of mitotic checkpoints in Saccharomyces cerevisiae. Mol Cell Biol. 1997 Jun;17(6):3315–3322. doi: 10.1128/mcb.17.6.3315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Taylor S. S., McKeon F. Kinetochore localization of murine Bub1 is required for normal mitotic timing and checkpoint response to spindle damage. Cell. 1997 May 30;89(5):727–735. doi: 10.1016/s0092-8674(00)80255-x. [DOI] [PubMed] [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. 1994 Dec;10(13):1793–1808. doi: 10.1002/yea.320101310. [DOI] [PubMed] [Google Scholar]
  34. Yu H. G., Muszynski M. G., Kelly Dawe R. The maize homologue of the cell cycle checkpoint protein MAD2 reveals kinetochore substructure and contrasting mitotic and meiotic localization patterns. J Cell Biol. 1999 May 3;145(3):425–435. doi: 10.1083/jcb.145.3.425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Zeng X., Kahana J. A., Silver P. A., Morphew M. K., McIntosh J. R., Fitch I. T., Carbon J., Saunders W. S. Slk19p is a centromere protein that functions to stabilize mitotic spindles. J Cell Biol. 1999 Jul 26;146(2):415–425. doi: 10.1083/jcb.146.2.415. [DOI] [PMC free article] [PubMed] [Google Scholar]

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