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. 1996 Apr 1;133(1):111–124. doi: 10.1083/jcb.133.1.111

Role of calmodulin and Spc110p interaction in the proper assembly of spindle pole body compenents

PMCID: PMC2120774  PMID: 8601600

Abstract

Previously we demonstrated that calmodulin binds to the carboxy terminus of Spc110p, an essential component of the Saccharomyces cerevisiae spindle pole body (SPB), and that this interaction is required for chromosome segregation. Immunoelectron microscopy presented here shows that calmodulin and thus the carboxy terminus of Spc110p localize to the central plaque. We created temperature- sensitive SPC110 mutations by combining PCR mutagenesis with a plasmid shuffle strategy. The temperature-sensitive allele spc110-220 differs from wild type at two sites. The cysteine 911 to arginine mutation resides in the calmodulin-binding site and alone confers a temperature- sensitive phenotype. Calmodulin overproduction suppresses the temperature sensitivity of spc110-220. Furthermore, calmodulin levels at the SPB decrease in the mutant cells at the restrictive temperature. Thus, calmodulin binding to Spc110-220p is defective at the nonpermissive temperature. Synchronized mutant cells incubated at the nonpermissive temperature arrest as large budded cells with a G2 content of DNA and suffer considerable lethality. Immunofluorescent staining demonstrates failure of nuclear DNA segregation and breakage of many spindles. Electron microscopy reveals an aberrant nuclear structure, the intranuclear microtubule organizer (IMO), that differs from a SPB but serves as a center of microtubule organization. The IMO appears during nascent SPB formation and disappears after SPB separation. The IMO contains both the 90-kD and the mutant 110-kD SPB components. Our results suggest that disruption of the calmodulin Spc110p interaction leads to the aberrant assembly of SPB components into the IMO, which in turn perturbs spindle formation.

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Selected References

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  1. Biggins S., Rose M. D. Direct interaction between yeast spindle pole body components: Kar1p is required for Cdc31p localization to the spindle pole body. J Cell Biol. 1994 May;125(4):843–852. doi: 10.1083/jcb.125.4.843. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Brockerhoff S. E., Davis T. N. Calmodulin concentrates at regions of cell growth in Saccharomyces cerevisiae. J Cell Biol. 1992 Aug;118(3):619–629. doi: 10.1083/jcb.118.3.619. [DOI] [PMC free article] [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. 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]
  6. Cadwell R. C., Joyce G. F. Randomization of genes by PCR mutagenesis. PCR Methods Appl. 1992 Aug;2(1):28–33. doi: 10.1101/gr.2.1.28. [DOI] [PubMed] [Google Scholar]
  7. Davidow L. S., Goetsch L., Byers B. Preferential Occurrence of Nonsister Spores in Two-Spored Asci of SACCHAROMYCES CEREVISIAE: Evidence for Regulation of Spore-Wall Formation by the Spindle Pole Body. Genetics. 1980 Mar;94(3):581–595. doi: 10.1093/genetics/94.3.581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Davis T. N., Thorner J. Vertebrate and yeast calmodulin, despite significant sequence divergence, are functionally interchangeable. Proc Natl Acad Sci U S A. 1989 Oct;86(20):7909–7913. doi: 10.1073/pnas.86.20.7909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. 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]
  13. 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]
  14. Ikura M., Clore G. M., Gronenborn A. M., Zhu G., Klee C. B., Bax A. Solution structure of a calmodulin-target peptide complex by multidimensional NMR. Science. 1992 May 1;256(5057):632–638. doi: 10.1126/science.1585175. [DOI] [PubMed] [Google Scholar]
  15. Ito M., Guerriero V., Jr, Chen X. M., Hartshorne D. J. Definition of the inhibitory domain of smooth muscle myosin light chain kinase by site-directed mutagenesis. Biochemistry. 1991 Apr 9;30(14):3498–3503. doi: 10.1021/bi00228a021. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Knighton D. R., Pearson R. B., Sowadski J. M., Means A. R., Ten Eyck L. F., Taylor S. S., Kemp B. E. Structural basis of the intrasteric regulation of myosin light chain kinases. Science. 1992 Oct 2;258(5079):130–135. doi: 10.1126/science.1439761. [DOI] [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. Lauzé E., Stoelcker B., Luca F. C., Weiss E., Schutz A. R., Winey M. Yeast spindle pole body duplication gene MPS1 encodes an essential dual specificity protein kinase. EMBO J. 1995 Apr 18;14(8):1655–1663. doi: 10.1002/j.1460-2075.1995.tb07154.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Meador W. E., Means A. R., Quiocho F. A. Modulation of calmodulin plasticity in molecular recognition on the basis of x-ray structures. Science. 1993 Dec 10;262(5140):1718–1721. doi: 10.1126/science.8259515. [DOI] [PubMed] [Google Scholar]
  22. Meador W. E., Means A. R., Quiocho F. A. Target enzyme recognition by calmodulin: 2.4 A structure of a calmodulin-peptide complex. Science. 1992 Aug 28;257(5074):1251–1255. doi: 10.1126/science.1519061. [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. Muhlrad D., Hunter R., Parker R. A rapid method for localized mutagenesis of yeast genes. Yeast. 1992 Feb;8(2):79–82. doi: 10.1002/yea.320080202. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. Roof D. M., Meluh P. B., Rose M. D. Kinesin-related proteins required for assembly of the mitotic spindle. J Cell Biol. 1992 Jul;118(1):95–108. doi: 10.1083/jcb.118.1.95. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Rose M. D., Fink G. R. KAR1, a gene required for function of both intranuclear and extranuclear microtubules in yeast. Cell. 1987 Mar 27;48(6):1047–1060. doi: 10.1016/0092-8674(87)90712-4. [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. Rout M. P., Kilmartin J. V. Yeast spindle pole body components. Cold Spring Harb Symp Quant Biol. 1991;56:687–692. doi: 10.1101/sqb.1991.056.01.077. [DOI] [PubMed] [Google Scholar]
  31. Schultz L. D. Transcriptional role of yeast deoxyribonucleic acid dependent ribonucleic acid polymerase III. Biochemistry. 1978 Feb 21;17(4):750–758. doi: 10.1021/bi00597a031. [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. Spang A., Courtney I., Fackler U., Matzner M., Schiebel E. The calcium-binding protein cell division cycle 31 of Saccharomyces cerevisiae is a component of the half bridge of the spindle pole body. J Cell Biol. 1993 Oct;123(2):405–416. doi: 10.1083/jcb.123.2.405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Spang A., Courtney I., Grein K., Matzner M., Schiebel E. The Cdc31p-binding protein Kar1p is a component of the half bridge of the yeast spindle pole body. J Cell Biol. 1995 Mar;128(5):863–877. doi: 10.1083/jcb.128.5.863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. Sullivan D. S., Huffaker T. C. Astral microtubules are not required for anaphase B in Saccharomyces cerevisiae. J Cell Biol. 1992 Oct;119(2):379–388. doi: 10.1083/jcb.119.2.379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Sun G. H., Hirata A., Ohya Y., Anraku Y. Mutations in yeast calmodulin cause defects in spindle pole body functions and nuclear integrity. J Cell Biol. 1992 Dec;119(6):1625–1639. doi: 10.1083/jcb.119.6.1625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Winey M., Goetsch L., Baum P., Byers B. MPS1 and MPS2: novel yeast genes defining distinct steps of spindle pole body duplication. J Cell Biol. 1991 Aug;114(4):745–754. doi: 10.1083/jcb.114.4.745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Winey M., Hoyt M. A., Chan C., Goetsch L., Botstein D., Byers B. NDC1: a nuclear periphery component required for yeast spindle pole body duplication. J Cell Biol. 1993 Aug;122(4):743–751. doi: 10.1083/jcb.122.4.743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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]
  41. Wright R., Rine J. Transmission electron microscopy and immunocytochemical studies of yeast: analysis of HMG-CoA reductase overproduction by electron microscopy. Methods Cell Biol. 1989;31:473–512. doi: 10.1016/s0091-679x(08)61624-6. [DOI] [PubMed] [Google Scholar]
  42. Zhu G., Muller E. G., Amacher S. L., Northrop J. L., Davis T. N. A dosage-dependent suppressor of a temperature-sensitive calmodulin mutant encodes a protein related to the fork head family of DNA-binding proteins. Mol Cell Biol. 1993 Mar;13(3):1779–1787. doi: 10.1128/mcb.13.3.1779. [DOI] [PMC free article] [PubMed] [Google Scholar]

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