Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1992 Aug 1;118(3):607–617. doi: 10.1083/jcb.118.3.607

A temperature-sensitive calmodulin mutant loses viability during mitosis

PMCID: PMC2289556  PMID: 1639846

Abstract

Although rare, a recessive temperature-sensitive calmodulin mutant has been isolated in Saccharomyces cerevisiae. The mutant carries two mutations in CMD1, isoleucine 100 is changed to asparagine and glutamic acid 104 is changed to valine. Neither mutation alone conferred temperature sensitivity. A single mutation that allowed production of an intact but defective protein was not identified. At the nonpermissive temperature, the temperature-sensitive mutant displayed multiple defects. Bud formation and growth was delayed, but this defect was not responsible for the temperature-sensitive lethality. Cells synchronized in G1 progressed through the cell cycle and retained viability until the movement of the nucleus to the neck between the mother cell and the large bud. After nuclear movement, less than 5% of the cells survived the first mitosis and could form colonies when returned to permissive conditions. The duplicated DNA was dispersed along the spindle, extending from mother to daughter cell. Cells synchronized in G2/M lost viability immediately upon the shift to the nonpermissive temperature. At a semipermissive temperature, the mutant showed approximately a 10-fold increase in the rate of chromosome loss compared to a wild-type strain. The mitotic phenotype is very similar to yeast mutants that are defective in chromosome disjunction. The mutant also showed defects in cytokinesis.

Full Text

The Full Text of this article is available as a PDF (1.8 MB).

Selected References

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

  1. Ackerman P., Glover C. V., Osheroff N. Phosphorylation of DNA topoisomerase II in vivo and in total homogenates of Drosophila Kc cells. The role of casein kinase II. J Biol Chem. 1988 Sep 5;263(25):12653–12660. [PubMed] [Google Scholar]
  2. Bachs O., Lanini L., Serratosa J., Coll M. J., Bastos R., Aligué R., Rius E., Carafoli E. Calmodulin-binding proteins in the nuclei of quiescent and proliferatively activated rat liver cells. J Biol Chem. 1990 Oct 25;265(30):18595–18600. [PubMed] [Google Scholar]
  3. Baitinger C., Alderton J., Poenie M., Schulman H., Steinhardt R. A. Multifunctional Ca2+/calmodulin-dependent protein kinase is necessary for nuclear envelope breakdown. J Cell Biol. 1990 Nov;111(5 Pt 1):1763–1773. doi: 10.1083/jcb.111.5.1763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Baum P., Furlong C., Byers B. Yeast gene required for spindle pole body duplication: homology of its product with Ca2+-binding proteins. Proc Natl Acad Sci U S A. 1986 Aug;83(15):5512–5516. doi: 10.1073/pnas.83.15.5512. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Baum P., Yip C., Goetsch L., Byers B. A yeast gene essential for regulation of spindle pole duplication. Mol Cell Biol. 1988 Dec;8(12):5386–5397. doi: 10.1128/mcb.8.12.5386. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Bond J. F., Fridovich-Keil J. L., Pillus L., Mulligan R. C., Solomon F. A chicken-yeast chimeric beta-tubulin protein is incorporated into mouse microtubules in vivo. Cell. 1986 Feb 14;44(3):461–468. doi: 10.1016/0092-8674(86)90467-8. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Brockerhoff S. E., Edmonds C. G., Davis T. N. Structural analysis of wild-type and mutant yeast calmodulins by limited proteolysis and electrospray ionization mass spectrometry. Protein Sci. 1992 Apr;1(4):504–516. doi: 10.1002/pro.5560010405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Coulondre C., Miller J. H. Genetic studies of the lac repressor. IV. Mutagenic specificity in the lacI gene of Escherichia coli. J Mol Biol. 1977 Dec 15;117(3):577–606. doi: 10.1016/0022-2836(77)90059-6. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Davis T. N., Urdea M. S., Masiarz F. R., Thorner J. Isolation of the yeast calmodulin gene: calmodulin is an essential protein. Cell. 1986 Nov 7;47(3):423–431. doi: 10.1016/0092-8674(86)90599-4. [DOI] [PubMed] [Google Scholar]
  13. Dinsmore J. H., Sloboda R. D. Calcium and calmodulin-dependent phosphorylation of a 62 kd protein induces microtubule depolymerization in sea urchin mitotic apparatuses. Cell. 1988 Jun 3;53(5):769–780. doi: 10.1016/0092-8674(88)90094-3. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Gimble F. S., Sauer R. T. Mutations in bacteriophage lambda repressor that prevent RecA-mediated cleavage. J Bacteriol. 1985 Apr;162(1):147–154. doi: 10.1128/jb.162.1.147-154.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hagan I., Yanagida M. Novel potential mitotic motor protein encoded by the fission yeast cut7+ gene. Nature. 1990 Oct 11;347(6293):563–566. doi: 10.1038/347563a0. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Hawthorne D C, Mortimer R K. Chromosome Mapping in Saccharomyces: Centromere-Linked Genes. Genetics. 1960 Aug;45(8):1085–1110. doi: 10.1093/genetics/45.8.1085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Holm C., Goto T., Wang J. C., Botstein D. DNA topoisomerase II is required at the time of mitosis in yeast. Cell. 1985 Jun;41(2):553–563. doi: 10.1016/s0092-8674(85)80028-3. [DOI] [PubMed] [Google Scholar]
  20. Holm C., Stearns T., Botstein D. DNA topoisomerase II must act at mitosis to prevent nondisjunction and chromosome breakage. Mol Cell Biol. 1989 Jan;9(1):159–168. doi: 10.1128/mcb.9.1.159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Howe C. L., Mooseker M. S. Characterization of the 110-kdalton actin-calmodulin-, and membrane-binding protein from microvilli of intestinal epithelial cells. J Cell Biol. 1983 Oct;97(4):974–985. doi: 10.1083/jcb.97.4.974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. Kao J. P., Alderton J. M., Tsien R. Y., Steinhardt R. A. Active involvement of Ca2+ in mitotic progression of Swiss 3T3 fibroblasts. J Cell Biol. 1990 Jul;111(1):183–196. doi: 10.1083/jcb.111.1.183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Koshland D., Kent J. C., Hartwell L. H. Genetic analysis of the mitotic transmission of minichromosomes. Cell. 1985 Feb;40(2):393–403. doi: 10.1016/0092-8674(85)90153-9. [DOI] [PubMed] [Google Scholar]
  26. Langer P. J., Shanabruch W. G., Walker G. C. Functional organization of plasmid pKM101. J Bacteriol. 1981 Mar;145(3):1310–1316. doi: 10.1128/jb.145.3.1310-1316.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. LeClerc J. E., Christensen J. R., Tata P. V., Christensen R. B., Lawrence C. W. Ultraviolet light induces different spectra of lacI sequence changes in vegetative and conjugating cells of Escherichia coli. J Mol Biol. 1988 Oct 5;203(3):619–633. doi: 10.1016/0022-2836(88)90197-0. [DOI] [PubMed] [Google Scholar]
  28. Levin D. E., Bartlett-Heubusch E. Mutants in the S. cerevisiae PKC1 gene display a cell cycle-specific osmotic stability defect. J Cell Biol. 1992 Mar;116(5):1221–1229. doi: 10.1083/jcb.116.5.1221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Ohkura H., Kinoshita N., Miyatani S., Toda T., Yanagida M. The fission yeast dis2+ gene required for chromosome disjoining encodes one of two putative type 1 protein phosphatases. Cell. 1989 Jun 16;57(6):997–1007. doi: 10.1016/0092-8674(89)90338-3. [DOI] [PubMed] [Google Scholar]
  31. Ohya Y., Anraku Y. A galactose-dependent cmd1 mutant of Saccharomyces cerevisiae: involvement of calmodulin in nuclear division. Curr Genet. 1989 Feb;15(2):113–120. doi: 10.1007/BF00435457. [DOI] [PubMed] [Google Scholar]
  32. Ohya Y., Anraku Y. Functional expression of chicken calmodulin in yeast. Biochem Biophys Res Commun. 1989 Jan 31;158(2):541–547. doi: 10.1016/s0006-291x(89)80083-x. [DOI] [PubMed] [Google Scholar]
  33. Palmer R. E., Koval M., Koshland D. The dynamics of chromosome movement in the budding yeast Saccharomyces cerevisiae. J Cell Biol. 1989 Dec;109(6 Pt 2):3355–3366. doi: 10.1083/jcb.109.6.3355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Persechini A., Kretsinger R. H., Davis T. N. Calmodulins with deletions in the central helix functionally replace the native protein in yeast cells. Proc Natl Acad Sci U S A. 1991 Jan 15;88(2):449–452. doi: 10.1073/pnas.88.2.449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. 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]
  37. Rasmussen C. D., Means A. R. Calmodulin is required for cell-cycle progression during G1 and mitosis. EMBO J. 1989 Jan;8(1):73–82. doi: 10.1002/j.1460-2075.1989.tb03350.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Rasmussen C. D., Means R. L., Lu K. P., May G. S., Means A. R. Characterization and expression of the unique calmodulin gene of Aspergillus nidulans. J Biol Chem. 1990 Aug 15;265(23):13767–13775. [PubMed] [Google Scholar]
  39. Runge K. W., Wellinger R. J., Zakian V. A. Effects of excess centromeres and excess telomeres on chromosome loss rates. Mol Cell Biol. 1991 Jun;11(6):2919–2928. doi: 10.1128/mcb.11.6.2919. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sahyoun N., Wolf M., Besterman J., Hsieh T., Sander M., LeVine H., 3rd, Chang K. J., Cuatrecasas P. Protein kinase C phosphorylates topoisomerase II: topoisomerase activation and its possible role in phorbol ester-induced differentiation of HL-60 cells. Proc Natl Acad Sci U S A. 1986 Mar;83(6):1603–1607. doi: 10.1073/pnas.83.6.1603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Schultz M. C., Reeder R. H., Hahn S. Variants of the TATA-binding protein can distinguish subsets of RNA polymerase I, II, and III promoters. Cell. 1992 May 15;69(4):697–702. doi: 10.1016/0092-8674(92)90233-3. [DOI] [PubMed] [Google Scholar]
  42. Sobue K., Muramoto Y., Fujita M., Kakiuchi S. Purification of a calmodulin-binding protein from chicken gizzard that interacts with F-actin. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5652–5655. doi: 10.1073/pnas.78.9.5652. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Staben C., Rabinowitz J. C. Nucleotide sequence of the Saccharomyces cerevisiae ADE3 gene encoding C1-tetrahydrofolate synthase. J Biol Chem. 1986 Apr 5;261(10):4629–4637. [PubMed] [Google Scholar]
  44. Sun G. H., Ohya Y., Anraku Y. Half-calmodulin is sufficient for cell proliferation. Expressions of N- and C-terminal halves of calmodulin in the yeast Saccharomyces cerevisiae. J Biol Chem. 1991 Apr 15;266(11):7008–7015. [PubMed] [Google Scholar]
  45. Sweet S. C., Rogers C. M., Welsh M. J. Calmodulin stabilization of kinetochore microtubule structure to the effect of nocodazole. J Cell Biol. 1988 Dec;107(6 Pt 1):2243–2251. doi: 10.1083/jcb.107.6.2243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Takeda T., Yamamoto M. Analysis and in vivo disruption of the gene coding for calmodulin in Schizosaccharomyces pombe. Proc Natl Acad Sci U S A. 1987 Jun;84(11):3580–3584. doi: 10.1073/pnas.84.11.3580. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Tanaka T., Kadowaki K., Lazarides E., Sobue K. Ca2(+)-dependent regulation of the spectrin/actin interaction by calmodulin and protein 4.1. J Biol Chem. 1991 Jan 15;266(2):1134–1140. [PubMed] [Google Scholar]
  48. Thomas J. H., Botstein D. A gene required for the separation of chromosomes on the spindle apparatus in yeast. Cell. 1986 Jan 17;44(1):65–76. doi: 10.1016/0092-8674(86)90485-x. [DOI] [PubMed] [Google Scholar]
  49. Todd P. A., Glickman B. W. Mutational specificity of UV light in Escherichia coli: indications for a role of DNA secondary structure. Proc Natl Acad Sci U S A. 1982 Jul;79(13):4123–4127. doi: 10.1073/pnas.79.13.4123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Uemura T., Tanagida M. Mitotic spindle pulls but fails to separate chromosomes in type II DNA topoisomerase mutants: uncoordinated mitosis. EMBO J. 1986 May;5(5):1003–1010. doi: 10.1002/j.1460-2075.1986.tb04315.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Uzawa S., Samejima I., Hirano T., Tanaka K., Yanagida M. The fission yeast cut1+ gene regulates spindle pole body duplication and has homology to the budding yeast ESP1 gene. Cell. 1990 Sep 7;62(5):913–925. doi: 10.1016/0092-8674(90)90266-h. [DOI] [PubMed] [Google Scholar]
  52. Vantard M., Lambert A. M., De Mey J., Picquot P., Van Eldik L. J. Characterization and immunocytochemical distribution of calmodulin in higher plant endosperm cells: localization in the mitotic apparatus. J Cell Biol. 1985 Aug;101(2):488–499. doi: 10.1083/jcb.101.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Walker G. C. Plasmid (pKM101)-mediated enhancement of repair and mutagenesis: dependence on chromosomal genes in Escherichia coli K-12. Mol Gen Genet. 1977 Mar 28;152(1):93–103. doi: 10.1007/BF00264945. [DOI] [PubMed] [Google Scholar]
  54. Wallis J. W., Chrebet G., Brodsky G., Rolfe M., Rothstein R. A hyper-recombination mutation in S. cerevisiae identifies a novel eukaryotic topoisomerase. Cell. 1989 Jul 28;58(2):409–419. doi: 10.1016/0092-8674(89)90855-6. [DOI] [PubMed] [Google Scholar]
  55. Welsh M. J., Dedman J. R., Brinkley B. R., Means A. R. Calcium-dependent regulator protein: localization in mitotic apparatus of eukaryotic cells. Proc Natl Acad Sci U S A. 1978 Apr;75(4):1867–1871. doi: 10.1073/pnas.75.4.1867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Welsh M. J., Dedman J. R., Brinkley B. R., Means A. R. Tubulin and calmodulin. Effects of microtubule and microfilament inhibitors on localization in the mitotic apparatus. J Cell Biol. 1979 Jun;81(3):624–634. doi: 10.1083/jcb.81.3.624. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. 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]
  58. Wood R. D., Skopek T. R., Hutchinson F. Changes in DNA base sequence induced by targeted mutagenesis of lambda phage by ultraviolet light. J Mol Biol. 1984 Mar 5;173(3):273–291. doi: 10.1016/0022-2836(84)90121-9. [DOI] [PubMed] [Google Scholar]
  59. Youderian P., Susskind M. M. Identification of the products of bacteriophage P22 genes, including a new late gene. Virology. 1980 Nov;107(1):258–269. doi: 10.1016/0042-6822(80)90291-3. [DOI] [PubMed] [Google Scholar]

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

RESOURCES