Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1993 Dec 1;123(5):1175–1184. doi: 10.1083/jcb.123.5.1175

A spacer protein in the Saccharomyces cerevisiae spindle poly body whose transcript is cell cycle-regulated

PMCID: PMC2119877  PMID: 7503995

Abstract

Monoclonal antibodies against the 110-kD component of the yeast spindle pole body (SPB) were used to clone the corresponding gene SPC110. SPC110 is identical to NUF1 (Mirzayan, C., C. S. Copeland, and M. Synder. 1992. J. Cell Biol. 116:1319-1332). SPC110/NUF1 has an MluI cell cycle box consensus sequence in its putative promoter region, and we found that the transcript was cell cycle regulated in a similar way to other MluI-regulated transcripts. Spc110p/Nuflp has a long central region with a predicted coiled-coil structure. We expressed this region in Escherichia coli and showed by rotary shadowing that rods of the predicted length were present. The 110-kD component is localized in the SPB to the gap between the central plaque and the sealed ends of the nuclear microtubules near the inner plaque (Rout, M., and J. V. Kilmartin. 1990. J. Cell Biol. 111:1913-1927). We found that rodlike structures bridge this gap. When truncations of SPC110 with deletions in the coiled-coil region of the protein replaced the wild-type gene, the gap between the central plaque and the ends of the microtubules decreased in proportion to the size of the deletion. This suggests that Spc110p connects these two parts of the SPB together and that the coiled-coil domain acts as a spacer element.

Full Text

The Full Text of this article is available as a PDF (2.6 MB).

Selected References

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

  1. Barker D. G., Johnston L. H. Saccharomyces cerevisiae cdc9, a structural gene for yeast DNA ligase which complements Schizosaccharomyces pombe cdc17. Eur J Biochem. 1983 Aug 1;134(2):315–319. doi: 10.1111/j.1432-1033.1983.tb07568.x. [DOI] [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. 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]
  4. 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]
  5. Byers B., Shriver K., Goetsch L. The role of spindle pole bodies and modified microtubule ends in the initiation of microtubule assembly in Saccharomyces cerevisiae. J Cell Sci. 1978 Apr;30:331–352. doi: 10.1242/jcs.30.1.331. [DOI] [PubMed] [Google Scholar]
  6. Cohen C., Parry D. A. Alpha-helical coiled coils and bundles: how to design an alpha-helical protein. Proteins. 1990;7(1):1–15. doi: 10.1002/prot.340070102. [DOI] [PubMed] [Google Scholar]
  7. Conde J., Fink G. R. A mutant of Saccharomyces cerevisiae defective for nuclear fusion. Proc Natl Acad Sci U S A. 1976 Oct;73(10):3651–3655. doi: 10.1073/pnas.73.10.3651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dirick L., Moll T., Auer H., Nasmyth K. A central role for SWI6 in modulating cell cycle Start-specific transcription in yeast. Nature. 1992 Jun 11;357(6378):508–513. doi: 10.1038/357508a0. [DOI] [PubMed] [Google Scholar]
  9. Furter R., Paravicini G., Aebi M., Braus G., Prantl F., Niederberger P., Hütter R. The TRP4 gene of Saccharomyces cerevisiae: isolation and structural analysis. Nucleic Acids Res. 1986 Aug 26;14(16):6357–6373. doi: 10.1093/nar/14.16.6357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Johnson A. L., Barker D. G., Johnston L. H. Induction of yeast DNA ligase genes in exponential and stationary phase cultures in response to DNA damaging agents. Curr Genet. 1986;11(2):107–112. doi: 10.1007/BF00378201. [DOI] [PubMed] [Google Scholar]
  12. Johnston L. H., Lowndes N. F., Johnson A. L., Sugino A. A cell-cycle-regulated trans-factor, DSC1, controls expression of DNA synthesis genes in yeast. Cold Spring Harb Symp Quant Biol. 1991;56:169–176. doi: 10.1101/sqb.1991.056.01.022. [DOI] [PubMed] [Google Scholar]
  13. Kassir Y., Kupiec M., Shalom A., Simchen G. Cloning and mapping of CDC40, a Saccharomyces cerevisiae gene with a role in DNA repair. Curr Genet. 1985;9(4):253–257. doi: 10.1007/BF00419952. [DOI] [PubMed] [Google Scholar]
  14. Katsura I. Determination of bacteriophage lambda tail length by a protein ruler. Nature. 1987 May 7;327(6117):73–75. doi: 10.1038/327073a0. [DOI] [PubMed] [Google Scholar]
  15. Katsura I., Hendrix R. W. Length determination in bacteriophage lambda tails. Cell. 1984 Dec;39(3 Pt 2):691–698. doi: 10.1016/0092-8674(84)90476-8. [DOI] [PubMed] [Google Scholar]
  16. Kilmartin J. V., Adams A. E. Structural rearrangements of tubulin and actin during the cell cycle of the yeast Saccharomyces. J Cell Biol. 1984 Mar;98(3):922–933. doi: 10.1083/jcb.98.3.922. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Lowndes N. F., Johnson A. L., Breeden L., Johnston L. H. SWI6 protein is required for transcription of the periodically expressed DNA synthesis genes in budding yeast. Nature. 1992 Jun 11;357(6378):505–508. doi: 10.1038/357505a0. [DOI] [PubMed] [Google Scholar]
  19. Marsh J. L., Erfle M., Wykes E. J. The pIC plasmid and phage vectors with versatile cloning sites for recombinant selection by insertional inactivation. Gene. 1984 Dec;32(3):481–485. doi: 10.1016/0378-1119(84)90022-2. [DOI] [PubMed] [Google Scholar]
  20. McIntosh E. M., Atkinson T., Storms R. K., Smith M. Characterization of a short, cis-acting DNA sequence which conveys cell cycle stage-dependent transcription in Saccharomyces cerevisiae. Mol Cell Biol. 1991 Jan;11(1):329–337. doi: 10.1128/mcb.11.1.329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Meuwissen R. L., Offenberg H. H., Dietrich A. J., Riesewijk A., van Iersel M., Heyting C. A coiled-coil related protein specific for synapsed regions of meiotic prophase chromosomes. EMBO J. 1992 Dec;11(13):5091–5100. doi: 10.1002/j.1460-2075.1992.tb05616.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Moens P. B., Rapport E. Spindles, spindle plaques, and meiosis in the yeast Saccharomyces cerevisiae (Hansen). J Cell Biol. 1971 Aug;50(2):344–361. doi: 10.1083/jcb.50.2.344. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Nagai K., Thøgersen H. C. Synthesis and sequence-specific proteolysis of hybrid proteins produced in Escherichia coli. Methods Enzymol. 1987;153:461–481. doi: 10.1016/0076-6879(87)53072-5. [DOI] [PubMed] [Google Scholar]
  25. Nasmyth K., Adolf G., Lydall D., Seddon A. The identification of a second cell cycle control on the HO promoter in yeast: cell cycle regulation of SW15 nuclear entry. Cell. 1990 Aug 24;62(4):631–647. doi: 10.1016/0092-8674(90)90110-z. [DOI] [PubMed] [Google Scholar]
  26. Nasmyth K. At least 1400 base pairs of 5'-flanking DNA is required for the correct expression of the HO gene in yeast. Cell. 1985 Aug;42(1):213–223. doi: 10.1016/s0092-8674(85)80117-3. [DOI] [PubMed] [Google Scholar]
  27. Page B. D., Snyder M. CIK1: a developmentally regulated spindle pole body-associated protein important for microtubule functions in Saccharomyces cerevisiae. Genes Dev. 1992 Aug;6(8):1414–1429. doi: 10.1101/gad.6.8.1414. [DOI] [PubMed] [Google Scholar]
  28. Pearson W. R., Lipman D. J. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. doi: 10.1073/pnas.85.8.2444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Peterson J. B., Gray R. H., Ris H. Meiotic spindle plaques in Saccharomyces cerevisiae. J Cell Biol. 1972 Jun;53(3):837–841. doi: 10.1083/jcb.53.3.837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Rothstein R. J. One-step gene disruption in yeast. Methods Enzymol. 1983;101:202–211. doi: 10.1016/0076-6879(83)01015-0. [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. 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]
  34. 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]
  35. Skehel J. M., Pilkington S. J., Runswick M. J., Fearnley I. M., Walker J. E. NADH:ubiquinone oxidoreductase from bovine heart mitochondria. Complementary DNA sequence of the import precursor of the 10 kDa subunit of the flavoprotein fragment. FEBS Lett. 1991 Apr 22;282(1):135–138. doi: 10.1016/0014-5793(91)80462-c. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  38. Sym M., Engebrecht J. A., Roeder G. S. ZIP1 is a synaptonemal complex protein required for meiotic chromosome synapsis. Cell. 1993 Feb 12;72(3):365–378. doi: 10.1016/0092-8674(93)90114-6. [DOI] [PubMed] [Google Scholar]
  39. Tyler J. M., Branton D. Rotary shadowing of extended molecules dried from glycerol. J Ultrastruct Res. 1980 May;71(2):95–102. doi: 10.1016/s0022-5320(80)90098-2. [DOI] [PubMed] [Google Scholar]
  40. Vallen E. A., Scherson T. Y., Roberts T., van Zee K., Rose M. D. Asymmetric mitotic segregation of the yeast spindle pole body. Cell. 1992 May 1;69(3):505–515. doi: 10.1016/0092-8674(92)90451-h. [DOI] [PubMed] [Google Scholar]
  41. Way M., Pope B., Gooch J., Hawkins M., Weeds A. G. Identification of a region in segment 1 of gelsolin critical for actin binding. EMBO J. 1990 Dec;9(12):4103–4109. doi: 10.1002/j.1460-2075.1990.tb07632.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Wehland J., Schröder H. C., Weber K. Amino acid sequence requirements in the epitope recognized by the alpha-tubulin-specific rat monoclonal antibody YL 1/2. EMBO J. 1984 Jun;3(6):1295–1300. doi: 10.1002/j.1460-2075.1984.tb01965.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wente S. R., Rout M. P., Blobel G. A new family of yeast nuclear pore complex proteins. J Cell Biol. 1992 Nov;119(4):705–723. doi: 10.1083/jcb.119.4.705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. White J. H., Barker D. G., Nurse P., Johnston L. H. Periodic transcription as a means of regulating gene expression during the cell cycle: contrasting modes of expression of DNA ligase genes in budding and fission yeast. EMBO J. 1986 Jul;5(7):1705–1709. doi: 10.1002/j.1460-2075.1986.tb04414.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. White J. H., Green S. R., Barker D. G., Dumas L. B., Johnston L. H. The CDC8 transcript is cell cycle regulated in yeast and is expressed coordinately with CDC9 and CDC21 at a point preceding histone transcription. Exp Cell Res. 1987 Jul;171(1):223–231. doi: 10.1016/0014-4827(87)90265-5. [DOI] [PubMed] [Google Scholar]
  46. 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]
  47. Young R. A., Davis R. W. Yeast RNA polymerase II genes: isolation with antibody probes. Science. 1983 Nov 18;222(4625):778–782. doi: 10.1126/science.6356359. [DOI] [PubMed] [Google Scholar]

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

RESOURCES