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. 1996 Mar 1;132(5):903–914. doi: 10.1083/jcb.132.5.903

The 110-kD spindle pole body component of Saccharomyces cerevisiae is a phosphoprotein that is modified in a cell cycle-dependent manner

PMCID: PMC2120732  PMID: 8603921

Abstract

Spc110p (Nuf1p) is an essential component of the yeast microtubule organizing center, or spindle pole body (SPB). Asynchronous wild-type cultures contain two electrophoretically distinct isoforms of Spc110p as detected by Western blot analysis, suggesting that Spc110p is modified in vivo. Both isoforms incorporate 32Pi in vivo, suggesting that Spc110p is post-translationally modified by phosphorylation. The slower-migrating 120-kD Spc110p isoform after incubation is converted to the faster-migrating 112-kD isoform after incubation with protein phosphatase PP2A, and specific PP2A inhibitors block this conversion. Thus, additional phosphorylation of Spc110p at serine and/or threonine residues gives rise to the slower-migrating 120-kD isoform. The 120-kD isoform predominates in cells arrested in mitosis by the addition of nocodazole. However, the 120-kD isoform is not detectable in cells grown to stationary phase (G0) or in cells arrested in G1 by the addition of alpha-factor. Temperature-sensitive cell division cycle (cdc) mutations demonstrate that the presence of the 120-kD isoform correlates with mitotic spindle formation but not with SPB duplication. In a synchronous wild-type population, the additional serine/threonine phosphorylation that gives rise to the 120-kD isoform appears as cells are forming the mitotic spindle and diminishes as cells enter anaphase. None of several sequences similar to the consensus for phosphorylation by the Cdc28p (cdc2p34) kinase is important for these mitosis-specific phosphorylations or for function. Carboxy-terminal Spc110p truncations lacking the calmodulin binding site can support growth and are also phosphorylated in a cell cycle-specific manner. Further truncation of the Spc110p carboxy terminus results in mutant proteins that are unable to support growth and now migrate as single species. Collectively, these results provide the first evidence of a structural component of the SPB that is phosphorylated during spindle formation and dephosphorylated as cells enter anaphase.

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Selected References

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  1. Bai C., Richman R., Elledge S. J. Human cyclin F. EMBO J. 1994 Dec 15;13(24):6087–6098. doi: 10.1002/j.1460-2075.1994.tb06955.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Black S., Andrews P. D., Sneddon A. A., Stark M. J. A regulated MET3-GLC7 gene fusion provides evidence of a mitotic role for Saccharomyces cerevisiae protein phosphatase 1. Yeast. 1995 Jun 30;11(8):747–759. doi: 10.1002/yea.320110806. [DOI] [PubMed] [Google Scholar]
  3. Boeke J. D., Trueheart J., Natsoulis G., Fink G. R. 5-Fluoroorotic acid as a selective agent in yeast molecular genetics. Methods Enzymol. 1987;154:164–175. doi: 10.1016/0076-6879(87)54076-9. [DOI] [PubMed] [Google Scholar]
  4. Byers B., Goetsch L. Behavior of spindles and spindle plaques in the cell cycle and conjugation of Saccharomyces cerevisiae. J Bacteriol. 1975 Oct;124(1):511–523. doi: 10.1128/jb.124.1.511-523.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Byers B., Goetsch L. Duplication of spindle plaques and integration of the yeast cell cycle. Cold Spring Harb Symp Quant Biol. 1974;38:123–131. doi: 10.1101/sqb.1974.038.01.016. [DOI] [PubMed] [Google Scholar]
  6. Byers B., Goetsch L. Preparation of yeast cells for thin-section electron microscopy. Methods Enzymol. 1991;194:602–608. doi: 10.1016/0076-6879(91)94044-d. [DOI] [PubMed] [Google Scholar]
  7. Davis T. N. A temperature-sensitive calmodulin mutant loses viability during mitosis. J Cell Biol. 1992 Aug;118(3):607–617. doi: 10.1083/jcb.118.3.607. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fitch I., Dahmann C., Surana U., Amon A., Nasmyth K., Goetsch L., Byers B., Futcher B. Characterization of four B-type cyclin genes of the budding yeast Saccharomyces cerevisiae. Mol Biol Cell. 1992 Jul;3(7):805–818. doi: 10.1091/mbc.3.7.805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Geiser J. R., Sundberg H. A., Chang B. H., Muller E. G., Davis T. N. The essential mitotic target of calmodulin is the 110-kilodalton component of the spindle pole body in Saccharomyces cerevisiae. Mol Cell Biol. 1993 Dec;13(12):7913–7924. doi: 10.1128/mcb.13.12.7913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Geiser J. R., van Tuinen D., Brockerhoff S. E., Neff M. M., Davis T. N. Can calmodulin function without binding calcium? Cell. 1991 Jun 14;65(6):949–959. doi: 10.1016/0092-8674(91)90547-c. [DOI] [PubMed] [Google Scholar]
  11. Glover D. M., Leibowitz M. H., McLean D. A., Parry H. Mutations in aurora prevent centrosome separation leading to the formation of monopolar spindles. Cell. 1995 Apr 7;81(1):95–105. doi: 10.1016/0092-8674(95)90374-7. [DOI] [PubMed] [Google Scholar]
  12. Golsteyn R. M., Mundt K. E., Fry A. M., Nigg E. A. Cell cycle regulation of the activity and subcellular localization of Plk1, a human protein kinase implicated in mitotic spindle function. J Cell Biol. 1995 Jun;129(6):1617–1628. doi: 10.1083/jcb.129.6.1617. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hartwell L. H., Smith D. Altered fidelity of mitotic chromosome transmission in cell cycle mutants of S. cerevisiae. Genetics. 1985 Jul;110(3):381–395. doi: 10.1093/genetics/110.3.381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hisamoto N., Sugimoto K., Matsumoto K. The Glc7 type 1 protein phosphatase of Saccharomyces cerevisiae is required for cell cycle progression in G2/M. Mol Cell Biol. 1994 May;14(5):3158–3165. doi: 10.1128/mcb.14.5.3158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jacobs C. W., Adams A. E., Szaniszlo P. J., Pringle J. R. Functions of microtubules in the Saccharomyces cerevisiae cell cycle. J Cell Biol. 1988 Oct;107(4):1409–1426. doi: 10.1083/jcb.107.4.1409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kilmartin J. V., Dyos S. L., Kershaw D., Finch J. T. A spacer protein in the Saccharomyces cerevisiae spindle poly body whose transcript is cell cycle-regulated. J Cell Biol. 1993 Dec;123(5):1175–1184. doi: 10.1083/jcb.123.5.1175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  19. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  20. Langan T. A., Gautier J., Lohka M., Hollingsworth R., Moreno S., Nurse P., Maller J., Sclafani R. A. Mammalian growth-associated H1 histone kinase: a homolog of cdc2+/CDC28 protein kinases controlling mitotic entry in yeast and frog cells. Mol Cell Biol. 1989 Sep;9(9):3860–3868. doi: 10.1128/mcb.9.9.3860. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. MacKelvie S. H., Andrews P. D., Stark M. J. The Saccharomyces cerevisiae gene SDS22 encodes a potential regulator of the mitotic function of yeast type 1 protein phosphatase. Mol Cell Biol. 1995 Jul;15(7):3777–3785. doi: 10.1128/mcb.15.7.3777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. McCarroll R. M., Fangman W. L. Time of replication of yeast centromeres and telomeres. Cell. 1988 Aug 12;54(4):505–513. doi: 10.1016/0092-8674(88)90072-4. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Muller E. G. Thioredoxin deficiency in yeast prolongs S phase and shortens the G1 interval of the cell cycle. J Biol Chem. 1991 May 15;266(14):9194–9202. [PubMed] [Google Scholar]
  25. Nasmyth K. Control of the yeast cell cycle by the Cdc28 protein kinase. Curr Opin Cell Biol. 1993 Apr;5(2):166–179. doi: 10.1016/0955-0674(93)90099-c. [DOI] [PubMed] [Google Scholar]
  26. Palmer R. E., Hogan E., Koshland D. Mitotic transmission of artificial chromosomes in cdc mutants of the yeast, Saccharomyces cerevisiae. Genetics. 1990 Aug;125(4):763–774. doi: 10.1093/genetics/125.4.763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Pringle J. R., Preston R. A., Adams A. E., Stearns T., Drubin D. G., Haarer B. K., Jones E. W. Fluorescence microscopy methods for yeast. Methods Cell Biol. 1989;31:357–435. doi: 10.1016/s0091-679x(08)61620-9. [DOI] [PubMed] [Google Scholar]
  28. Rose M. D., Biggins S., Satterwhite L. L. Unravelling the tangled web at the microtubule-organizing center. Curr Opin Cell Biol. 1993 Feb;5(1):105–115. doi: 10.1016/s0955-0674(05)80015-8. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Schwob E., Böhm T., Mendenhall M. D., Nasmyth K. The B-type cyclin kinase inhibitor p40SIC1 controls the G1 to S transition in S. cerevisiae. Cell. 1994 Oct 21;79(2):233–244. doi: 10.1016/0092-8674(94)90193-7. [DOI] [PubMed] [Google Scholar]
  31. Shenoy S., Choi J. K., Bagrodia S., Copeland T. D., Maller J. L., Shalloway D. Purified maturation promoting factor phosphorylates pp60c-src at the sites phosphorylated during fibroblast mitosis. Cell. 1989 Jun 2;57(5):763–774. doi: 10.1016/0092-8674(89)90791-5. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Slater M. L., Sharrow S. O., Gart J. J. Cell cycle of Saccharomycescerevisiae in populations growing at different rates. Proc Natl Acad Sci U S A. 1977 Sep;74(9):3850–3854. doi: 10.1073/pnas.74.9.3850. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Stirling D. A., Welch K. A., Stark M. J. Interaction with calmodulin is required for the function of Spc110p, an essential component of the yeast spindle pole body. EMBO J. 1994 Sep 15;13(18):4329–4342. doi: 10.1002/j.1460-2075.1994.tb06753.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Tabor S., Richardson C. C. DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4767–4771. doi: 10.1073/pnas.84.14.4767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Vandré D. D., Borisy G. G. Anaphase onset and dephosphorylation of mitotic phosphoproteins occur concomitantly. J Cell Sci. 1989 Oct;94(Pt 2):245–258. doi: 10.1242/jcs.94.2.245. [DOI] [PubMed] [Google Scholar]
  37. Vandré D. D., Wills V. L. Inhibition of mitosis by okadaic acid: possible involvement of a protein phosphatase 2A in the transition from metaphase to anaphase. J Cell Sci. 1992 Jan;101(Pt 1):79–91. doi: 10.1242/jcs.101.1.79. [DOI] [PubMed] [Google Scholar]
  38. Warner J. R. Labeling of RNA and phosphoproteins in Saccharomyces cerevisiae. Methods Enzymol. 1991;194:423–428. doi: 10.1016/0076-6879(91)94033-9. [DOI] [PubMed] [Google Scholar]
  39. Westendorf J. M., Rao P. N., Gerace L. Cloning of cDNAs for M-phase phosphoproteins recognized by the MPM2 monoclonal antibody and determination of the phosphorylated epitope. Proc Natl Acad Sci U S A. 1994 Jan 18;91(2):714–718. doi: 10.1073/pnas.91.2.714. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Winey M., Byers B. Assembly and functions of the spindle pole body in budding yeast. Trends Genet. 1993 Sep;9(9):300–304. doi: 10.1016/0168-9525(93)90247-f. [DOI] [PubMed] [Google Scholar]
  41. Wright A. P., Bruns M., Hartley B. S. Extraction and rapid inactivation of proteins from Saccharomyces cerevisiae by trichloroacetic acid precipitation. Yeast. 1989 Jan-Feb;5(1):51–53. doi: 10.1002/yea.320050107. [DOI] [PubMed] [Google Scholar]

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