Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1995 Nov 1;131(3):709–720. doi: 10.1083/jcb.131.3.709

Mad1p, a phosphoprotein component of the spindle assembly checkpoint in budding yeast

PMCID: PMC2120625  PMID: 7593191

Abstract

The spindle assembly checkpoint prevents cells from initiating anaphase until the spindle has been fully assembled. We previously isolated mitotic arrest deficient (mad) mutants that inactivate this checkpoint and thus increase the sensitivity of cells to benomyl, a drug that interferes with mitotic spindle assembly by depolymerizing microtubules. We have cloned the MAD1 gene and show that when it is disrupted yeast cells have the same phenotype as the previously isolated mad1 mutants: they fail to delay the metaphase to anaphase transition in response to microtubule depolymerization. MAD1 is predicted to encode a 90-kD coiled-coil protein. Anti-Mad1p antibodies give a novel punctate nuclear staining pattern and cell fractionation reveals that the bulk of Mad1p is soluble. Mad1p becomes hyperphosphorylated when wild-type cells are arrested in mitosis by benomyl treatment, or by placing a cold sensitive tubulin mutant at the restrictive temperature. This modification does not occur in G1- arrested cells treated with benomyl or in cells arrested in mitosis by defects in the mitotic cyclin proteolysis machinery, suggesting that Mad1p hyperphosphorylation is a step in the activation of the spindle assembly checkpoint. Analysis of Mad1p phosphorylation in other spindle assembly checkpoint mutants reveals that this response to microtubule- disrupting agents is defective in some (mad2, bub1, and bub3) but not all (mad3, bub2) mutant strains. We discuss the possible functions of Mad1p at this cell cycle checkpoint.

Full Text

The Full Text of this article is available as a PDF (2.5 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  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., Irniger S., Nasmyth K. Closing the cell cycle circle in yeast: G2 cyclin proteolysis initiated at mitosis persists until the activation of G1 cyclins in the next cycle. Cell. 1994 Jul 1;77(7):1037–1050. doi: 10.1016/0092-8674(94)90443-x. [DOI] [PubMed] [Google Scholar]
  3. Dingwall C., Laskey R. A. Nuclear targeting sequences--a consensus? Trends Biochem Sci. 1991 Dec;16(12):478–481. doi: 10.1016/0968-0004(91)90184-w. [DOI] [PubMed] [Google Scholar]
  4. Enoch T., Carr A. M., Nurse P. Fission yeast genes involved in coupling mitosis to completion of DNA replication. Genes Dev. 1992 Nov;6(11):2035–2046. doi: 10.1101/gad.6.11.2035. [DOI] [PubMed] [Google Scholar]
  5. Enoch T., Nurse P. Mutation of fission yeast cell cycle control genes abolishes dependence of mitosis on DNA replication. Cell. 1990 Feb 23;60(4):665–673. doi: 10.1016/0092-8674(90)90669-6. [DOI] [PubMed] [Google Scholar]
  6. Ford J. C., al-Khodairy F., Fotou E., Sheldrick K. S., Griffiths D. J., Carr A. M. 14-3-3 protein homologs required for the DNA damage checkpoint in fission yeast. Science. 1994 Jul 22;265(5171):533–535. doi: 10.1126/science.8036497. [DOI] [PubMed] [Google Scholar]
  7. Fuchs E., Weber K. Intermediate filaments: structure, dynamics, function, and disease. Annu Rev Biochem. 1994;63:345–382. doi: 10.1146/annurev.bi.63.070194.002021. [DOI] [PubMed] [Google Scholar]
  8. Futcher B., Carbon J. Toxic effects of excess cloned centromeres. Mol Cell Biol. 1986 Jun;6(6):2213–2222. doi: 10.1128/mcb.6.6.2213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hackney D. D., Levitt J. D., Suhan J. Kinesin undergoes a 9 S to 6 S conformational transition. J Biol Chem. 1992 Apr 25;267(12):8696–8701. [PubMed] [Google Scholar]
  10. Hartwell L. H., Weinert T. A. Checkpoints: controls that ensure the order of cell cycle events. Science. 1989 Nov 3;246(4930):629–634. doi: 10.1126/science.2683079. [DOI] [PubMed] [Google Scholar]
  11. Henikoff S. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene. 1984 Jun;28(3):351–359. doi: 10.1016/0378-1119(84)90153-7. [DOI] [PubMed] [Google Scholar]
  12. Hirano T., Mitchison T. J. A heterodimeric coiled-coil protein required for mitotic chromosome condensation in vitro. Cell. 1994 Nov 4;79(3):449–458. doi: 10.1016/0092-8674(94)90254-2. [DOI] [PubMed] [Google Scholar]
  13. Holloway S. L., Glotzer M., King R. W., Murray A. W. Anaphase is initiated by proteolysis rather than by the inactivation of maturation-promoting factor. Cell. 1993 Jul 2;73(7):1393–1402. doi: 10.1016/0092-8674(93)90364-v. [DOI] [PubMed] [Google Scholar]
  14. Hoyt M. A., Totis L., Roberts B. T. S. cerevisiae genes required for cell cycle arrest in response to loss of microtubule function. Cell. 1991 Aug 9;66(3):507–517. doi: 10.1016/0092-8674(81)90014-3. [DOI] [PubMed] [Google Scholar]
  15. Huffaker T. C., Thomas J. H., Botstein D. Diverse effects of beta-tubulin mutations on microtubule formation and function. J Cell Biol. 1988 Jun;106(6):1997–2010. doi: 10.1083/jcb.106.6.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hussain M., Lenard J. Characterization of PDR4, a Saccharomyces cerevisiae gene that confers pleiotropic drug resistance in high-copy number: identity with YAP1, encoding a transcriptional activator [corrected]. Gene. 1991 May 15;101(1):149–152. doi: 10.1016/0378-1119(91)90238-7. [DOI] [PubMed] [Google Scholar]
  17. Klein F., Laroche T., Cardenas M. E., Hofmann J. F., Schweizer D., Gasser S. M. Localization of RAP1 and topoisomerase II in nuclei and meiotic chromosomes of yeast. J Cell Biol. 1992 Jun;117(5):935–948. doi: 10.1083/jcb.117.5.935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Li R., Murray A. W. Feedback control of mitosis in budding yeast. Cell. 1991 Aug 9;66(3):519–531. doi: 10.1016/0092-8674(81)90015-5. [DOI] [PubMed] [Google Scholar]
  19. Lupas A., Van Dyke M., Stock J. Predicting coiled coils from protein sequences. Science. 1991 May 24;252(5009):1162–1164. doi: 10.1126/science.252.5009.1162. [DOI] [PubMed] [Google Scholar]
  20. Minshull J., Sun H., Tonks N. K., Murray A. W. A MAP kinase-dependent spindle assembly checkpoint in Xenopus egg extracts. Cell. 1994 Nov 4;79(3):475–486. doi: 10.1016/0092-8674(94)90256-9. [DOI] [PubMed] [Google Scholar]
  21. Mirzayan C., Copeland C. S., Snyder M. The NUF1 gene encodes an essential coiled-coil related protein that is a potential component of the yeast nucleoskeleton. J Cell Biol. 1992 Mar;116(6):1319–1332. doi: 10.1083/jcb.116.6.1319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Morgan D. O. Principles of CDK regulation. Nature. 1995 Mar 9;374(6518):131–134. doi: 10.1038/374131a0. [DOI] [PubMed] [Google Scholar]
  23. Moye-Rowley W. S., Harshman K. D., Parker C. S. Yeast YAP1 encodes a novel form of the jun family of transcriptional activator proteins. Genes Dev. 1989 Mar;3(3):283–292. doi: 10.1101/gad.3.3.283. [DOI] [PubMed] [Google Scholar]
  24. Murray A. W. The genetics of cell cycle checkpoints. Curr Opin Genet Dev. 1995 Feb;5(1):5–11. doi: 10.1016/s0959-437x(95)90046-2. [DOI] [PubMed] [Google Scholar]
  25. Murray A. Cell cycle checkpoints. Curr Opin Cell Biol. 1994 Dec;6(6):872–876. doi: 10.1016/0955-0674(94)90059-0. [DOI] [PubMed] [Google Scholar]
  26. Nakajima H., Hirata A., Ogawa Y., Yonehara T., Yoda K., Yamasaki M. A cytoskeleton-related gene, uso1, is required for intracellular protein transport in Saccharomyces cerevisiae. J Cell Biol. 1991 Apr;113(2):245–260. doi: 10.1083/jcb.113.2.245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Norbury C., Nurse P. Animal cell cycles and their control. Annu Rev Biochem. 1992;61:441–470. doi: 10.1146/annurev.bi.61.070192.002301. [DOI] [PubMed] [Google Scholar]
  28. Philippsen P., Stotz A., Scherf C. DNA of Saccharomyces cerevisiae. Methods Enzymol. 1991;194:169–182. doi: 10.1016/0076-6879(91)94014-4. [DOI] [PubMed] [Google Scholar]
  29. Rieder C. L., Schultz A., Cole R., Sluder G. Anaphase onset in vertebrate somatic cells is controlled by a checkpoint that monitors sister kinetochore attachment to the spindle. J Cell Biol. 1994 Dec;127(5):1301–1310. doi: 10.1083/jcb.127.5.1301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Roberts B. T., Farr K. A., Hoyt M. A. The Saccharomyces cerevisiae checkpoint gene BUB1 encodes a novel protein kinase. Mol Cell Biol. 1994 Dec;14(12):8282–8291. doi: 10.1128/mcb.14.12.8282. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Rose M. D., Novick P., Thomas J. H., Botstein D., Fink G. R. A Saccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector. Gene. 1987;60(2-3):237–243. doi: 10.1016/0378-1119(87)90232-0. [DOI] [PubMed] [Google Scholar]
  32. Rout M. P., Kilmartin J. V. Components of the yeast spindle and spindle pole body. J Cell Biol. 1990 Nov;111(5 Pt 1):1913–1927. doi: 10.1083/jcb.111.5.1913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rowley R., Subramani S., Young P. G. Checkpoint controls in Schizosaccharomyces pombe: rad1. EMBO J. 1992 Apr;11(4):1335–1342. doi: 10.1002/j.1460-2075.1992.tb05178.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rubin G. M. Preparation of RNA and ribosomes from yeast. Methods Cell Biol. 1975;12:45–64. doi: 10.1016/s0091-679x(08)60951-6. [DOI] [PubMed] [Google Scholar]
  35. Schnell N., Krems B., Entian K. D. The PAR1 (YAP1/SNQ3) gene of Saccharomyces cerevisiae, a c-jun homologue, is involved in oxygen metabolism. Curr Genet. 1992 Apr;21(4-5):269–273. doi: 10.1007/BF00351681. [DOI] [PubMed] [Google Scholar]
  36. Sikorski R. S., Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19–27. doi: 10.1093/genetics/122.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Smith D. B., Johnson K. S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. doi: 10.1016/0378-1119(88)90005-4. [DOI] [PubMed] [Google Scholar]
  38. Spencer F., Hieter P. Centromere DNA mutations induce a mitotic delay in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1992 Oct 1;89(19):8908–8912. doi: 10.1073/pnas.89.19.8908. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Stearns T., Hoyt M. A., Botstein D. Yeast mutants sensitive to antimicrotubule drugs define three genes that affect microtubule function. Genetics. 1990 Feb;124(2):251–262. doi: 10.1093/genetics/124.2.251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Surana U., Amon A., Dowzer C., McGrew J., Byers B., Nasmyth K. Destruction of the CDC28/CLB mitotic kinase is not required for the metaphase to anaphase transition in budding yeast. EMBO J. 1993 May;12(5):1969–1978. doi: 10.1002/j.1460-2075.1993.tb05846.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Ward G. E., Kirschner M. W. Identification of cell cycle-regulated phosphorylation sites on nuclear lamin C. Cell. 1990 May 18;61(4):561–577. doi: 10.1016/0092-8674(90)90469-u. [DOI] [PubMed] [Google Scholar]
  42. Weinert T. A., Hartwell L. H. The RAD9 gene controls the cell cycle response to DNA damage in Saccharomyces cerevisiae. Science. 1988 Jul 15;241(4863):317–322. doi: 10.1126/science.3291120. [DOI] [PubMed] [Google Scholar]
  43. Weinert T. A., Kiser G. L., Hartwell L. H. Mitotic checkpoint genes in budding yeast and the dependence of mitosis on DNA replication and repair. Genes Dev. 1994 Mar 15;8(6):652–665. doi: 10.1101/gad.8.6.652. [DOI] [PubMed] [Google Scholar]
  44. Wu A., Wemmie J. A., Edgington N. P., Goebl M., Guevara J. L., Moye-Rowley W. S. Yeast bZip proteins mediate pleiotropic drug and metal resistance. J Biol Chem. 1993 Sep 5;268(25):18850–18858. [PubMed] [Google Scholar]
  45. Yang C. H., Lambie E. J., Snyder M. NuMA: an unusually long coiled-coil related protein in the mammalian nucleus. J Cell Biol. 1992 Mar;116(6):1303–1317. doi: 10.1083/jcb.116.6.1303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Yen T. J., Li G., Schaar B. T., Szilak I., Cleveland D. W. CENP-E is a putative kinetochore motor that accumulates just before mitosis. Nature. 1992 Oct 8;359(6395):536–539. doi: 10.1038/359536a0. [DOI] [PubMed] [Google Scholar]
  47. al-Khodairy F., Carr A. M. DNA repair mutants defining G2 checkpoint pathways in Schizosaccharomyces pombe. EMBO J. 1992 Apr;11(4):1343–1350. doi: 10.1002/j.1460-2075.1992.tb05179.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. al-Khodairy F., Enoch T., Hagan I. M., Carr A. M. The Schizosaccharomyces pombe hus5 gene encodes a ubiquitin conjugating enzyme required for normal mitosis. J Cell Sci. 1995 Feb;108(Pt 2):475–486. doi: 10.1242/jcs.108.2.475. [DOI] [PubMed] [Google Scholar]
  49. al-Khodairy F., Fotou E., Sheldrick K. S., Griffiths D. J., Lehmann A. R., Carr A. M. Identification and characterization of new elements involved in checkpoint and feedback controls in fission yeast. Mol Biol Cell. 1994 Feb;5(2):147–160. doi: 10.1091/mbc.5.2.147. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES