Factors required for the binding of reassembled yeast kinetochores to microtubules in vitro (original) (raw)

Abstract

Kinetochores are structures that assemble on centromeric DNA and mediate the attachment of chromosomes to the microtubules of the mitotic spindle. The protein components of kinetochores are poorly understood, but the simplicity of the S. cerevisiae kinetochore makes it an attractive candidate for molecular dissection. Mutations in genes encoding CBF1 and CBF3, proteins that bind to yeast centromeres, interfere with chromosome segregation in vivo. To determine the roles played by these factors and by various regions of centromeric DNA in kinetochore function, we have developed a method to partially reassemble kinetochores on exogenous centromeric templates in vitro and to visualize the attachment of these reassembled kinetochore complexes to microtubules. In this assay, single reassembled complexes appear to mediate microtubule binding. We find that CBF3 is absolutely essential for this attachment but, contrary to previous reports (Hyman, A. A., K. Middleton, M. Centola, T.J. Mitchison, and J. Carbon. 1992. Microtubule- motor activity of a yeast centromere-binding protein complex. Nature (Lond.). 359:533-536) is not sufficient. Additional cellular factors interact with CBF3 to form active microtubule-binding complexes. This is mediated primarily by the CDEIII region of centromeric DNA but CDEII plays an essential modulatory role. Thus, the attachment of kinetochores to microtubules appears to involve a hierarchy of interactions by factors that assemble on a core complex consisting of DNA-bound CBF3.

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Selected References

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  1. Bernat R. L., Borisy G. G., Rothfield N. F., Earnshaw W. C. Injection of anticentromere antibodies in interphase disrupts events required for chromosome movement at mitosis. J Cell Biol. 1990 Oct;111(4):1519–1533. doi: 10.1083/jcb.111.4.1519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Blanton S., Srinivasan A., Rymond B. C. PRP38 encodes a yeast protein required for pre-mRNA splicing and maintenance of stable U6 small nuclear RNA levels. Mol Cell Biol. 1992 Sep;12(9):3939–3947. doi: 10.1128/mcb.12.9.3939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bloom K. S., Carbon J. Yeast centromere DNA is in a unique and highly ordered structure in chromosomes and small circular minichromosomes. Cell. 1982 Jun;29(2):305–317. doi: 10.1016/0092-8674(82)90147-7. [DOI] [PubMed] [Google Scholar]
  4. Brown J. D., Beggs J. D. Roles of PRP8 protein in the assembly of splicing complexes. EMBO J. 1992 Oct;11(10):3721–3729. doi: 10.1002/j.1460-2075.1992.tb05457.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cai M., Davis R. W. Yeast centromere binding protein CBF1, of the helix-loop-helix protein family, is required for chromosome stability and methionine prototrophy. Cell. 1990 May 4;61(3):437–446. doi: 10.1016/0092-8674(90)90525-j. [DOI] [PubMed] [Google Scholar]
  6. Clarke L. Centromeres of budding and fission yeasts. Trends Genet. 1990 May;6(5):150–154. doi: 10.1016/0168-9525(90)90149-z. [DOI] [PubMed] [Google Scholar]
  7. Compton D. A., Yen T. J., Cleveland D. W. Identification of novel centromere/kinetochore-associated proteins using monoclonal antibodies generated against human mitotic chromosome scaffolds. J Cell Biol. 1991 Mar;112(6):1083–1097. doi: 10.1083/jcb.112.6.1083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Earnshaw W. C., Tomkiel J. E. Centromere and kinetochore structure. Curr Opin Cell Biol. 1992 Feb;4(1):86–93. doi: 10.1016/0955-0674(92)90063-i. [DOI] [PubMed] [Google Scholar]
  10. Fitzgerald-Hayes M., Clarke L., Carbon J. Nucleotide sequence comparisons and functional analysis of yeast centromere DNAs. Cell. 1982 May;29(1):235–244. doi: 10.1016/0092-8674(82)90108-8. [DOI] [PubMed] [Google Scholar]
  11. Gaudet A., Fitzgerald-Hayes M. Alterations in the adenine-plus-thymine-rich region of CEN3 affect centromere function in Saccharomyces cerevisiae. Mol Cell Biol. 1987 Jan;7(1):68–75. doi: 10.1128/mcb.7.1.68. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Goh P. Y., Kilmartin J. V. NDC10: a gene involved in chromosome segregation in Saccharomyces cerevisiae. J Cell Biol. 1993 May;121(3):503–512. doi: 10.1083/jcb.121.3.503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hegemann J. H., Shero J. H., Cottarel G., Philippsen P., Hieter P. Mutational analysis of centromere DNA from chromosome VI of Saccharomyces cerevisiae. Mol Cell Biol. 1988 Jun;8(6):2523–2535. doi: 10.1128/mcb.8.6.2523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Howard J., Hyman A. A. Preparation of marked microtubules for the assay of the polarity of microtubule-based motors by fluorescence microscopy. Methods Cell Biol. 1993;39:105–113. doi: 10.1016/s0091-679x(08)60164-8. [DOI] [PubMed] [Google Scholar]
  15. Hoyt M. A., He L., Loo K. K., Saunders W. S. Two Saccharomyces cerevisiae kinesin-related gene products required for mitotic spindle assembly. J Cell Biol. 1992 Jul;118(1):109–120. doi: 10.1083/jcb.118.1.109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hyman A. A., Middleton K., Centola M., Mitchison T. J., Carbon J. Microtubule-motor activity of a yeast centromere-binding protein complex. Nature. 1992 Oct 8;359(6395):533–536. doi: 10.1038/359533a0. [DOI] [PubMed] [Google Scholar]
  17. Hyman A. A., Mitchison T. J. Two different microtubule-based motor activities with opposite polarities in kinetochores. Nature. 1991 May 16;351(6323):206–211. doi: 10.1038/351206a0. [DOI] [PubMed] [Google Scholar]
  18. Jiang W., Lechner J., Carbon J. Isolation and characterization of a gene (CBF2) specifying a protein component of the budding yeast kinetochore. J Cell Biol. 1993 May;121(3):513–519. doi: 10.1083/jcb.121.3.513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jiang W., Middleton K., Yoon H. J., Fouquet C., Carbon J. An essential yeast protein, CBF5p, binds in vitro to centromeres and microtubules. Mol Cell Biol. 1993 Aug;13(8):4884–4893. doi: 10.1128/mcb.13.8.4884. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kingsbury J., Koshland D. Centromere function on minichromosomes isolated from budding yeast. Mol Biol Cell. 1993 Aug;4(8):859–870. doi: 10.1091/mbc.4.8.859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kingsbury J., Koshland D. Centromere-dependent binding of yeast minichromosomes to microtubules in vitro. Cell. 1991 Aug 9;66(3):483–495. doi: 10.1016/0092-8674(81)90012-x. [DOI] [PubMed] [Google Scholar]
  22. Lechner J., Carbon J. A 240 kd multisubunit protein complex, CBF3, is a major component of the budding yeast centromere. Cell. 1991 Feb 22;64(4):717–725. doi: 10.1016/0092-8674(91)90501-o. [DOI] [PubMed] [Google Scholar]
  23. McGrew J., Diehl B., Fitzgerald-Hayes M. Single base-pair mutations in centromere element III cause aberrant chromosome segregation in Saccharomyces cerevisiae. Mol Cell Biol. 1986 Feb;6(2):530–538. doi: 10.1128/mcb.6.2.530. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. McIntosh J. R., Pfarr C. M. Mitotic motors. J Cell Biol. 1991 Nov;115(3):577–585. doi: 10.1083/jcb.115.3.577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mellor J., Jiang W., Funk M., Rathjen J., Barnes C. A., Hinz T., Hegemann J. H., Philippsen P. CPF1, a yeast protein which functions in centromeres and promoters. EMBO J. 1990 Dec;9(12):4017–4026. doi: 10.1002/j.1460-2075.1990.tb07623.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Meluh P. B., Rose M. D. KAR3, a kinesin-related gene required for yeast nuclear fusion. Cell. 1990 Mar 23;60(6):1029–1041. doi: 10.1016/0092-8674(90)90351-e. [DOI] [PubMed] [Google Scholar]
  27. Mitchison T. J., Kirschner M. W. Properties of the kinetochore in vitro. II. Microtubule capture and ATP-dependent translocation. J Cell Biol. 1985 Sep;101(3):766–777. doi: 10.1083/jcb.101.3.766. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mitchison T. J. Microtubule dynamics and kinetochore function in mitosis. Annu Rev Cell Biol. 1988;4:527–549. doi: 10.1146/annurev.cb.04.110188.002523. [DOI] [PubMed] [Google Scholar]
  29. Murphy M. R., Fowlkes D. M., Fitzgerald-Hayes M. Analysis of centromere function in Saccharomyces cerevisiae using synthetic centromere mutants. Chromosoma. 1991 Dec;101(3):189–197. doi: 10.1007/BF00355368. [DOI] [PubMed] [Google Scholar]
  30. Ng R., Carbon J. Mutational and in vitro protein-binding studies on centromere DNA from Saccharomyces cerevisiae. Mol Cell Biol. 1987 Dec;7(12):4522–4534. doi: 10.1128/mcb.7.12.4522. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Niedenthal R., Stoll R., Hegemann J. H. In vivo characterization of the Saccharomyces cerevisiae centromere DNA element I, a binding site for the helix-loop-helix protein CPF1. Mol Cell Biol. 1991 Jul;11(7):3545–3553. doi: 10.1128/mcb.11.7.3545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Peterson J. B., Ris H. Electron-microscopic study of the spindle and chromosome movement in the yeast Saccharomyces cerevisiae. J Cell Sci. 1976 Nov;22(2):219–242. doi: 10.1242/jcs.22.2.219. [DOI] [PubMed] [Google Scholar]
  33. Pfarr C. M., Coue M., Grissom P. M., Hays T. S., Porter M. E., McIntosh J. R. Cytoplasmic dynein is localized to kinetochores during mitosis. Nature. 1990 May 17;345(6272):263–265. doi: 10.1038/345263a0. [DOI] [PubMed] [Google Scholar]
  34. Saunders W. S., Hoyt M. A. Kinesin-related proteins required for structural integrity of the mitotic spindle. Cell. 1992 Aug 7;70(3):451–458. doi: 10.1016/0092-8674(92)90169-d. [DOI] [PubMed] [Google Scholar]
  35. Sawin K. E., Scholey J. M. Motor proteins in cell division. Trends Cell Biol. 1991 Nov;1(5):122–129. doi: 10.1016/0962-8924(91)90117-r. [DOI] [PubMed] [Google Scholar]
  36. Schatz P. J., Pillus L., Grisafi P., Solomon F., Botstein D. Two functional alpha-tubulin genes of the yeast Saccharomyces cerevisiae encode divergent proteins. Mol Cell Biol. 1986 Nov;6(11):3711–3721. doi: 10.1128/mcb.6.11.3711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Schulman I. G., Bloom K. Genetic dissection of centromere function. Mol Cell Biol. 1993 Jun;13(6):3156–3166. doi: 10.1128/mcb.13.6.3156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Skibbens R. V., Skeen V. P., Salmon E. D. Directional instability of kinetochore motility during chromosome congression and segregation in mitotic newt lung cells: a push-pull mechanism. J Cell Biol. 1993 Aug;122(4):859–875. doi: 10.1083/jcb.122.4.859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Spencer F., Gerring S. L., Connelly C., Hieter P. Mitotic chromosome transmission fidelity mutants in Saccharomyces cerevisiae. Genetics. 1990 Feb;124(2):237–249. doi: 10.1093/genetics/124.2.237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Yen T. J., Compton D. A., Wise D., Zinkowski R. P., Brinkley B. R., Earnshaw W. C., Cleveland D. W. CENP-E, a novel human centromere-associated protein required for progression from metaphase to anaphase. EMBO J. 1991 May;10(5):1245–1254. doi: 10.1002/j.1460-2075.1991.tb08066.x. [DOI] [PMC free article] [PubMed] [Google Scholar]