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. 1992 Jan;3(1):29–47. doi: 10.1091/mbc.3.1.29

Yeast proteins associated with microtubules in vitro and in vivo.

G Barnes 1, K A Louie 1, D Botstein 1
PMCID: PMC275500  PMID: 1348005

Abstract

Conditions were established for the self-assembly of milligram amounts of purified Saccharomyces cerevisiae tubulin. Microtubules assembled with pure yeast tubulin were not stabilized by taxol; hybrid microtubules containing substoichiometric amounts of bovine tubulin were stabilized. Yeast microtubule-associated proteins (MAPs) were identified on affinity matrices made from hybrid and all-bovine microtubules. About 25 yeast MAPs were isolated. The amino-terminal sequences of several of these were determined: three were known metabolic enzymes, two were GTP-binding proteins (including the product of the SAR1 gene), and three were novel proteins not found in sequence databases. Affinity-purified antisera were generated against synthetic peptides corresponding to two of the apparently novel proteins (38 and 50 kDa). Immunofluorescence microscopy showed that both these proteins colocalize with intra- and extranuclear microtubules in vivo.

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Selected References

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

  1. Adams A. E., Pringle J. R. Relationship of actin and tubulin distribution to bud growth in wild-type and morphogenetic-mutant Saccharomyces cerevisiae. J Cell Biol. 1984 Mar;98(3):934–945. doi: 10.1083/jcb.98.3.934. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Amos L. A., Baker T. S. The three-dimensional structure of tubulin protofilaments. Nature. 1979 Jun 14;279(5714):607–612. doi: 10.1038/279607a0. [DOI] [PubMed] [Google Scholar]
  3. Barnes G., Drubin D. G., Stearns T. The cytoskeleton of Saccharomyces cerevisiae. Curr Opin Cell Biol. 1990 Feb;2(1):109–115. doi: 10.1016/s0955-0674(05)80040-7. [DOI] [PubMed] [Google Scholar]
  4. Basson M. E., Moore R. L., O'Rear J., Rine J. Identifying mutations in duplicated functions in Saccharomyces cerevisiae: recessive mutations in HMG-CoA reductase genes. Genetics. 1987 Dec;117(4):645–655. doi: 10.1093/genetics/117.4.645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Berlin V., Styles C. A., Fink G. R. BIK1, a protein required for microtubule function during mating and mitosis in Saccharomyces cerevisiae, colocalizes with tubulin. J Cell Biol. 1990 Dec;111(6 Pt 1):2573–2586. doi: 10.1083/jcb.111.6.2573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. 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]
  9. Drubin D. G., Miller K. G., Botstein D. Yeast actin-binding proteins: evidence for a role in morphogenesis. J Cell Biol. 1988 Dec;107(6 Pt 2):2551–2561. doi: 10.1083/jcb.107.6.2551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Duncan K., Edwards R. M., Coggins J. R. The pentafunctional arom enzyme of Saccharomyces cerevisiae is a mosaic of monofunctional domains. Biochem J. 1987 Sep 1;246(2):375–386. doi: 10.1042/bj2460375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Evans L., Mitchison T., Kirschner M. Influence of the centrosome on the structure of nucleated microtubules. J Cell Biol. 1985 Apr;100(4):1185–1191. doi: 10.1083/jcb.100.4.1185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gibbons I. R., Gibbons B. H., Mocz G., Asai D. J. Multiple nucleotide-binding sites in the sequence of dynein beta heavy chain. Nature. 1991 Aug 15;352(6336):640–643. doi: 10.1038/352640a0. [DOI] [PubMed] [Google Scholar]
  13. Hendriks W., Mulders J. W., Bibby M. A., Slingsby C., Bloemendal H., de Jong W. W. Duck lens epsilon-crystallin and lactate dehydrogenase B4 are identical: a single-copy gene product with two distinct functions. Proc Natl Acad Sci U S A. 1988 Oct;85(19):7114–7118. doi: 10.1073/pnas.85.19.7114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hoyt M. A., Stearns T., Botstein D. Chromosome instability mutants of Saccharomyces cerevisiae that are defective in microtubule-mediated processes. Mol Cell Biol. 1990 Jan;10(1):223–234. doi: 10.1128/mcb.10.1.223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Huitorel P., Pantaloni D. Bundling of microtubules by glyceraldehyde-3-phosphate dehydrogenase and its modulation by ATP. Eur J Biochem. 1985 Jul 15;150(2):265–269. doi: 10.1111/j.1432-1033.1985.tb09016.x. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Kataoka T., Powers S., McGill C., Fasano O., Strathern J., Broach J., Wigler M. Genetic analysis of yeast RAS1 and RAS2 genes. Cell. 1984 Jun;37(2):437–445. doi: 10.1016/0092-8674(84)90374-x. [DOI] [PubMed] [Google Scholar]
  19. Kellogg D. R., Field C. M., Alberts B. M. Identification of microtubule-associated proteins in the centrosome, spindle, and kinetochore of the early Drosophila embryo. J Cell Biol. 1989 Dec;109(6 Pt 1):2977–2991. doi: 10.1083/jcb.109.6.2977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kelly R. B. Associations between microtubules and intracellular organelles. Curr Opin Cell Biol. 1990 Feb;2(1):105–108. doi: 10.1016/s0955-0674(05)80039-0. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Kilmartin J. V. Purification of yeast tubulin by self-assembly in vitro. Biochemistry. 1981 Jun 9;20(12):3629–3633. doi: 10.1021/bi00515a050. [DOI] [PubMed] [Google Scholar]
  23. Kilmartin J. V., Wright B., Milstein C. Rat monoclonal antitubulin antibodies derived by using a new nonsecreting rat cell line. J Cell Biol. 1982 Jun;93(3):576–582. doi: 10.1083/jcb.93.3.576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kumagai H., Sakai H. A porcine brain protein (35 K protein) which bundles microtubules and its identification as glyceraldehyde 3-phosphate dehydrogenase. J Biochem. 1983 May;93(5):1259–1269. doi: 10.1093/oxfordjournals.jbchem.a134260. [DOI] [PubMed] [Google Scholar]
  25. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  26. 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]
  27. Lapetina E. G., Reep B. R. Specific binding of [alpha-32P]GTP to cytosolic and membrane-bound proteins of human platelets correlates with the activation of phospholipase C. Proc Natl Acad Sci U S A. 1987 Apr;84(8):2261–2265. doi: 10.1073/pnas.84.8.2261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lataste H., Senilh V., Wright M., Guénard D., Potier P. Relationships between the structures of taxol and baccatine III derivatives and their in vitro action on the disassembly of mammalian brain and Physarum amoebal microtubules. Proc Natl Acad Sci U S A. 1984 Jul;81(13):4090–4094. doi: 10.1073/pnas.81.13.4090. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lee G., Cowan N., Kirschner M. The primary structure and heterogeneity of tau protein from mouse brain. Science. 1988 Jan 15;239(4837):285–288. doi: 10.1126/science.3122323. [DOI] [PubMed] [Google Scholar]
  30. Lewis S. A., Wang D. H., Cowan N. J. Microtubule-associated protein MAP2 shares a microtubule binding motif with tau protein. Science. 1988 Nov 11;242(4880):936–939. doi: 10.1126/science.3142041. [DOI] [PubMed] [Google Scholar]
  31. Lippincott-Schwartz J., Donaldson J. G., Schweizer A., Berger E. G., Hauri H. P., Yuan L. C., Klausner R. D. Microtubule-dependent retrograde transport of proteins into the ER in the presence of brefeldin A suggests an ER recycling pathway. Cell. 1990 Mar 9;60(5):821–836. doi: 10.1016/0092-8674(90)90096-w. [DOI] [PubMed] [Google Scholar]
  32. Makarow M. Secretion of invertase in mitotic yeast cells. EMBO J. 1988 May;7(5):1475–1482. doi: 10.1002/j.1460-2075.1988.tb02965.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. McAlister L., Holland M. J. Differential expression of the three yeast glyceraldehyde-3-phosphate dehydrogenase genes. J Biol Chem. 1985 Dec 5;260(28):15019–15027. [PubMed] [Google Scholar]
  34. McAlister L., Holland M. J. Isolation and characterization of yeast strains carrying mutations in the glyceraldehyde-3-phosphate dehydrogenase genes. J Biol Chem. 1985 Dec 5;260(28):15013–15018. [PubMed] [Google Scholar]
  35. Meluh P. B., Rose M. D. KAR3, a kinesin-related gene required for yeast nuclear fusion. Cell. 1990 Mar 23;60(6):1029–1041. doi: 10.1016/0092-8674(90)90351-e. [DOI] [PubMed] [Google Scholar]
  36. Mitchison T., Kirschner M. Dynamic instability of microtubule growth. Nature. 1984 Nov 15;312(5991):237–242. doi: 10.1038/312237a0. [DOI] [PubMed] [Google Scholar]
  37. Mitchison T., Kirschner M. Microtubule assembly nucleated by isolated centrosomes. Nature. 1984 Nov 15;312(5991):232–237. doi: 10.1038/312232a0. [DOI] [PubMed] [Google Scholar]
  38. Nakańo A., Muramatsu M. A novel GTP-binding protein, Sar1p, is involved in transport from the endoplasmic reticulum to the Golgi apparatus. J Cell Biol. 1989 Dec;109(6 Pt 1):2677–2691. doi: 10.1083/jcb.109.6.2677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Neff N. F., Thomas J. H., Grisafi P., Botstein D. Isolation of the beta-tubulin gene from yeast and demonstration of its essential function in vivo. Cell. 1983 May;33(1):211–219. doi: 10.1016/0092-8674(83)90350-1. [DOI] [PubMed] [Google Scholar]
  40. Noble M., Lewis S. A., Cowan N. J. The microtubule binding domain of microtubule-associated protein MAP1B contains a repeated sequence motif unrelated to that of MAP2 and tau. J Cell Biol. 1989 Dec;109(6 Pt 2):3367–3376. doi: 10.1083/jcb.109.6.3367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  42. Obar R. A., Collins C. A., Hammarback J. A., Shpetner H. S., Vallee R. B. Molecular cloning of the microtubule-associated mechanochemical enzyme dynamin reveals homology with a new family of GTP-binding proteins. Nature. 1990 Sep 20;347(6290):256–261. doi: 10.1038/347256a0. [DOI] [PubMed] [Google Scholar]
  43. Ogawa K. Four ATP-binding sites in the midregion of the beta heavy chain of dynein. Nature. 1991 Aug 15;352(6336):643–645. doi: 10.1038/352643a0. [DOI] [PubMed] [Google Scholar]
  44. Ohta K., Toriyama M., Miyazaki M., Murofushi H., Hosoda S., Endo S., Sakai H. The mitotic apparatus-associated 51-kDa protein from sea urchin eggs is a GTP-binding protein and is immunologically related to yeast polypeptide elongation factor 1 alpha. J Biol Chem. 1990 Feb 25;265(6):3240–3247. [PubMed] [Google Scholar]
  45. Olmsted J. B. Microtubule-associated proteins. Annu Rev Cell Biol. 1986;2:421–457. doi: 10.1146/annurev.cb.02.110186.002225. [DOI] [PubMed] [Google Scholar]
  46. Peterson G. L. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem. 1977 Dec;83(2):346–356. doi: 10.1016/0003-2697(77)90043-4. [DOI] [PubMed] [Google Scholar]
  47. 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]
  48. 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]
  49. Rose M. D., Misra L. M., Vogel J. P. KAR2, a karyogamy gene, is the yeast homolog of the mammalian BiP/GRP78 gene. Cell. 1989 Jun 30;57(7):1211–1221. doi: 10.1016/0092-8674(89)90058-5. [DOI] [PubMed] [Google Scholar]
  50. Rosenkrantz M., Alam T., Kim K. S., Clark B. J., Srere P. A., Guarente L. P. Mitochondrial and nonmitochondrial citrate synthases in Saccharomyces cerevisiae are encoded by distinct homologous genes. Mol Cell Biol. 1986 Dec;6(12):4509–4515. doi: 10.1128/mcb.6.12.4509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Schatz P. J., Pillus L., Grisafi P., Solomon F., Botstein D. Two functional alpha-tubulin genes of the yeast Saccharomyces cerevisiae encode divergent proteins. Mol Cell Biol. 1986 Nov;6(11):3711–3721. doi: 10.1128/mcb.6.11.3711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. 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]
  53. Schatz P. J., Solomon F., Botstein D. Isolation and characterization of conditional-lethal mutations in the TUB1 alpha-tubulin gene of the yeast Saccharomyces cerevisiae. Genetics. 1988 Nov;120(3):681–695. doi: 10.1093/genetics/120.3.681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Schiff P. B., Fant J., Horwitz S. B. Promotion of microtubule assembly in vitro by taxol. Nature. 1979 Feb 22;277(5698):665–667. doi: 10.1038/277665a0. [DOI] [PubMed] [Google Scholar]
  55. Schulze E., Kirschner M. Dynamic and stable populations of microtubules in cells. J Cell Biol. 1987 Feb;104(2):277–288. doi: 10.1083/jcb.104.2.277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Segev N., Mulholland J., Botstein D. The yeast GTP-binding YPT1 protein and a mammalian counterpart are associated with the secretion machinery. Cell. 1988 Mar 25;52(6):915–924. doi: 10.1016/0092-8674(88)90433-3. [DOI] [PubMed] [Google Scholar]
  57. 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]
  58. Stearns T., Hoyt M. A., Botstein D. Yeast mutants sensitive to antimicrotubule drugs define three genes that affect microtubule function. Genetics. 1990 Feb;124(2):251–262. doi: 10.1093/genetics/124.2.251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Stearns T., Kahn R. A., Botstein D., Hoyt M. A. ADP ribosylation factor is an essential protein in Saccharomyces cerevisiae and is encoded by two genes. Mol Cell Biol. 1990 Dec;10(12):6690–6699. doi: 10.1128/mcb.10.12.6690. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Thomas J. H., Neff N. F., Botstein D. Isolation and characterization of mutations in the beta-tubulin gene of Saccharomyces cerevisiae. Genetics. 1985 Dec;111(4):715–734. doi: 10.1093/genetics/111.4.715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Vallee R. B., Collins C. A. Purification of microtubules and microtubule-associated proteins from sea urchin eggs and cultured mammalian cells using taxol, and use of exogenous taxol-stabilized brain microtubules for purifying microtubule-associated proteins. Methods Enzymol. 1986;134:116–127. doi: 10.1016/0076-6879(86)34080-1. [DOI] [PubMed] [Google Scholar]
  62. Wistow G., Piatigorsky J. Recruitment of enzymes as lens structural proteins. Science. 1987 Jun 19;236(4808):1554–1556. doi: 10.1126/science.3589669. [DOI] [PubMed] [Google Scholar]
  63. Yang J. T., Laymon R. A., Goldstein L. S. A three-domain structure of kinesin heavy chain revealed by DNA sequence and microtubule binding analyses. Cell. 1989 Mar 10;56(5):879–889. doi: 10.1016/0092-8674(89)90692-2. [DOI] [PubMed] [Google Scholar]
  64. Zhou R. P., Oskarsson M., Paules R. S., Schulz N., Cleveland D., Vande Woude G. F. Ability of the c-mos product to associate with and phosphorylate tubulin. Science. 1991 Feb 8;251(4994):671–675. doi: 10.1126/science.1825142. [DOI] [PubMed] [Google Scholar]

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