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. 1995 Nov 2;131(4):1003–1014. doi: 10.1083/jcb.131.4.1003

Yeast Num1p associates with the mother cell cortex during S/G2 phase and affects microtubular functions

PMCID: PMC2200000  PMID: 7490278

Abstract

The NUM1 gene is involved in the control of nuclear migration in Saccharomyces cerevisiae. The content of NUM1 mRNA fluctuates during the cell cycle, reaching a maximum at S/G2 phase, and the translation product Num1p associates with the cortex of mother cells mainly during S, G2, and mitosis, as seen by indirect immunofluorescence. The nuclear spindle in NUM1-deficient large-budded cells often fails to align along the mother/bud axis, while abnormally elongated astral microtubules emanate from both spindle pole bodies. A num1 null mutation confers temperature sensitivity to the cold-sensitive alpha-tubulin mutant tub1- 1, and shows synthetic lethality with the beta-tubulin mutant alleles tub2-402, tub2-403, tub2-404, and tub2-405. Deletion mapping has defined three functionally important Num1p regions: a potential EF hand Ca2+ binding site, a cluster of potential phosphorylation sites and a pleckstrin homology domain. The latter domain appears to be involved in targeting Num1p to the mother cell cortex. Our data suggest that the periodically expressed NUM1 gene product controls nuclear migration by affecting astral microtubule functions.

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Selected References

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  1. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Clark S. W., Meyer D. I. ACT3: a putative centractin homologue in S. cerevisiae is required for proper orientation of the mitotic spindle. J Cell Biol. 1994 Oct;127(1):129–138. doi: 10.1083/jcb.127.1.129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Eshel D., Urrestarazu L. A., Vissers S., Jauniaux J. C., van Vliet-Reedijk J. C., Planta R. J., Gibbons I. R. Cytoplasmic dynein is required for normal nuclear segregation in yeast. Proc Natl Acad Sci U S A. 1993 Dec 1;90(23):11172–11176. doi: 10.1073/pnas.90.23.11172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Fischer R., Timberlake W. E. Aspergillus nidulans apsA (anucleate primary sterigmata) encodes a coiled-coil protein required for nuclear positioning and completion of asexual development. J Cell Biol. 1995 Feb;128(4):485–498. doi: 10.1083/jcb.128.4.485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gibson T. J., Hyvönen M., Musacchio A., Saraste M., Birney E. PH domain: the first anniversary. Trends Biochem Sci. 1994 Sep;19(9):349–353. doi: 10.1016/0968-0004(94)90108-2. [DOI] [PubMed] [Google Scholar]
  7. Harlan J. E., Hajduk P. J., Yoon H. S., Fesik S. W. Pleckstrin homology domains bind to phosphatidylinositol-4,5-bisphosphate. Nature. 1994 Sep 8;371(6493):168–170. doi: 10.1038/371168a0. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. 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]
  11. Kormanec J., Schaaff-Gerstenschläger I., Zimmermann F. K., Perecko D., Küntzel H. Nuclear migration in Saccharomyces cerevisiae is controlled by the highly repetitive 313 kDa NUM1 protein. Mol Gen Genet. 1991 Nov;230(1-2):277–287. doi: 10.1007/BF00290678. [DOI] [PubMed] [Google Scholar]
  12. Küntzel H., Rottjakob H. W., Schwed A., Zwerschke W. START control in cycling Saccharomyces cerevisiae cells. Prog Nucleic Acid Res Mol Biol. 1994;48:1–28. doi: 10.1016/s0079-6603(08)60851-x. [DOI] [PubMed] [Google Scholar]
  13. Li Y. Y., Yeh E., Hays T., Bloom K. Disruption of mitotic spindle orientation in a yeast dynein mutant. Proc Natl Acad Sci U S A. 1993 Nov 1;90(21):10096–10100. doi: 10.1073/pnas.90.21.10096. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. McMillan J. N., Tatchell K. The JNM1 gene in the yeast Saccharomyces cerevisiae is required for nuclear migration and spindle orientation during the mitotic cell cycle. J Cell Biol. 1994 Apr;125(1):143–158. doi: 10.1083/jcb.125.1.143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Muhua L., Karpova T. S., Cooper J. A. A yeast actin-related protein homologous to that in vertebrate dynactin complex is important for spindle orientation and nuclear migration. Cell. 1994 Aug 26;78(4):669–679. doi: 10.1016/0092-8674(94)90531-2. [DOI] [PubMed] [Google Scholar]
  16. Munder T., Mink M., Küntzel H. Domains of the Saccharomyces cerevisiae CDC25 gene controlling mitosis and meiosis. Mol Gen Genet. 1988 Oct;214(2):271–277. doi: 10.1007/BF00337721. [DOI] [PubMed] [Google Scholar]
  17. Musacchio A., Gibson T., Rice P., Thompson J., Saraste M. The PH domain: a common piece in the structural patchwork of signalling proteins. Trends Biochem Sci. 1993 Sep;18(9):343–348. doi: 10.1016/0968-0004(93)90071-t. [DOI] [PubMed] [Google Scholar]
  18. Osley M. A. The regulation of histone synthesis in the cell cycle. Annu Rev Biochem. 1991;60:827–861. doi: 10.1146/annurev.bi.60.070191.004143. [DOI] [PubMed] [Google Scholar]
  19. Palmer R. E., Sullivan D. S., Huffaker T., Koshland D. Role of astral microtubules and actin in spindle orientation and migration in the budding yeast, Saccharomyces cerevisiae. J Cell Biol. 1992 Nov;119(3):583–593. doi: 10.1083/jcb.119.3.583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Pringle J. R., Adams A. E., Drubin D. G., Haarer B. K. Immunofluorescence methods for yeast. Methods Enzymol. 1991;194:565–602. doi: 10.1016/0076-6879(91)94043-c. [DOI] [PubMed] [Google Scholar]
  21. Repetto B., Tzagoloff A. Structure and regulation of KGD2, the structural gene for yeast dihydrolipoyl transsuccinylase. Mol Cell Biol. 1990 Aug;10(8):4221–4232. doi: 10.1128/mcb.10.8.4221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Revardel E., Aigle M. The NUM1 yeast gene: length polymorphism and physiological aspects of mutant phenotype. Yeast. 1993 May;9(5):495–506. doi: 10.1002/yea.320090505. [DOI] [PubMed] [Google Scholar]
  23. Richardson H., Lew D. J., Henze M., Sugimoto K., Reed S. I. Cyclin-B homologs in Saccharomyces cerevisiae function in S phase and in G2. Genes Dev. 1992 Nov;6(11):2021–2034. doi: 10.1101/gad.6.11.2021. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Schatz P. J., Solomon F., Botstein D. Genetically essential and nonessential alpha-tubulin genes specify functionally interchangeable proteins. Mol Cell Biol. 1986 Nov;6(11):3722–3733. doi: 10.1128/mcb.6.11.3722. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Schwob E., Nasmyth K. CLB5 and CLB6, a new pair of B cyclins involved in DNA replication in Saccharomyces cerevisiae. Genes Dev. 1993 Jul;7(7A):1160–1175. doi: 10.1101/gad.7.7a.1160. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Smith D. E., Fisher P. A. Identification, developmental regulation, and response to heat shock of two antigenically related forms of a major nuclear envelope protein in Drosophila embryos: application of an improved method for affinity purification of antibodies using polypeptides immobilized on nitrocellulose blots. J Cell Biol. 1984 Jul;99(1 Pt 1):20–28. doi: 10.1083/jcb.99.1.20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Stearns T., Botstein D. Unlinked noncomplementation: isolation of new conditional-lethal mutations in each of the tubulin genes of Saccharomyces cerevisiae. Genetics. 1988 Jun;119(2):249–260. doi: 10.1093/genetics/119.2.249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. 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]
  32. Welch M. D., Holtzman D. A., Drubin D. G. The yeast actin cytoskeleton. Curr Opin Cell Biol. 1994 Feb;6(1):110–119. doi: 10.1016/0955-0674(94)90124-4. [DOI] [PubMed] [Google Scholar]
  33. Zwerschke W., Rottjakob H. W., Küntzel H. The Saccharomyces cerevisiae CDC6 gene is transcribed at late mitosis and encodes a ATP/GTPase controlling S phase initiation. J Biol Chem. 1994 Sep 16;269(37):23351–23356. [PubMed] [Google Scholar]

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