Skip to main content
NCBI home page
The EMBO Journal logoLink to The EMBO Journal
. 1998 Feb 16;17(4):952–966. doi: 10.1093/emboj/17.4.952

A novel protein complex promoting formation of functional alpha- and gamma-tubulin.

S Geissler 1, K Siegers 1, E Schiebel 1
PMCID: PMC1170445  PMID: 9463374

Abstract

We describe the identification of GIM1/YKE2, GIM2/PAC10, GIM3, GIM4 and GIM5 in a screen for mutants that are synthetically lethal with tub4-1, encoding a mutated yeast gamma-tubulin. The cytoplasmic Gim proteins encoded by these GIM genes are present in common complexes as judged by co-immunoprecipitation and gel filtration experiments. The disruption of any of these genes results in similar phenotypes: the gim null mutants are synthetically lethal with tub4-1 and super-sensitive towards the microtubule-depolymerizing drug benomyl. All except Deltagim4 are cold-sensitive and their microtubules disassemble at 14 degrees C. The Gim proteins have one function related to alpha-tubulin and another to Tub4p, supported by the finding that the benomyl super-sensitivity is caused by a reduced level of alpha-tubulin while the synthetic lethality with tub4-1 is not. In addition, GIM1/YKE2 genetically interacts with two distinct classes of genes, one of which is involved in tubulin folding and the other in microtubule nucleation. We show that the Gim proteins are important for Tub4p function and bind to overproduced Tub4p. The mammalian homologues of GIM1/YKE2 and GIM2/PAC10 rescue the synthetically lethal phenotype with tub4-1 as well as the cold-sensitivity and benomyl super-sensitivity of the yeast deletion mutants. We suggest that the Gim proteins form a protein complex that promotes formation of functional alpha- and gamma-tubulin.

Full Text

The Full Text of this article is available as a PDF (564.5 KB).

Selected References

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

  1. Abe K., Wei J. F., Wei F. S., Hsu Y. C., Uehara H., Artzt K., Bennett D. Searching for coding sequences in the mammalian genome: the H-2K region of the mouse MHC is replete with genes expressed in embryos. EMBO J. 1988 Nov;7(11):3441–3449. doi: 10.1002/j.1460-2075.1988.tb03218.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Akashi T., Yoon Y., Oakley B. R. Characterization of gamma-tubulin complexes in Aspergillus nidulans and detection of putative gamma-tubulin interacting proteins. Cell Motil Cytoskeleton. 1997;37(2):149–158. doi: 10.1002/(SICI)1097-0169(1997)37:2<149::AID-CM7>3.0.CO;2-3. [DOI] [PubMed] [Google Scholar]
  3. Archer J. E., Vega L. R., Solomon F. Rbl2p, a yeast protein that binds to beta-tubulin and participates in microtubule function in vivo. Cell. 1995 Aug 11;82(3):425–434. doi: 10.1016/0092-8674(95)90431-x. [DOI] [PubMed] [Google Scholar]
  4. Ayscough K. R., Stryker J., Pokala N., Sanders M., Crews P., Drubin D. G. High rates of actin filament turnover in budding yeast and roles for actin in establishment and maintenance of cell polarity revealed using the actin inhibitor latrunculin-A. J Cell Biol. 1997 Apr 21;137(2):399–416. doi: 10.1083/jcb.137.2.399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  6. Burke D., Gasdaska P., Hartwell L. Dominant effects of tubulin overexpression in Saccharomyces cerevisiae. Mol Cell Biol. 1989 Mar;9(3):1049–1059. doi: 10.1128/mcb.9.3.1049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Chen X., Sullivan D. S., Huffaker T. C. Two yeast genes with similarity to TCP-1 are required for microtubule and actin function in vivo. Proc Natl Acad Sci U S A. 1994 Sep 13;91(19):9111–9115. doi: 10.1073/pnas.91.19.9111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Christianson T. W., Sikorski R. S., Dante M., Shero J. H., Hieter P. Multifunctional yeast high-copy-number shuttle vectors. Gene. 1992 Jan 2;110(1):119–122. doi: 10.1016/0378-1119(92)90454-w. [DOI] [PubMed] [Google Scholar]
  10. Drubin D. G. Actin and actin-binding proteins in yeast. Cell Motil Cytoskeleton. 1990;15(1):7–11. doi: 10.1002/cm.970150103. [DOI] [PubMed] [Google Scholar]
  11. Felsenstein J. Inferring phylogenies from protein sequences by parsimony, distance, and likelihood methods. Methods Enzymol. 1996;266:418–427. doi: 10.1016/s0076-6879(96)66026-1. [DOI] [PubMed] [Google Scholar]
  12. Fields S., Song O. A novel genetic system to detect protein-protein interactions. Nature. 1989 Jul 20;340(6230):245–246. doi: 10.1038/340245a0. [DOI] [PubMed] [Google Scholar]
  13. Frydman J., Nimmesgern E., Erdjument-Bromage H., Wall J. S., Tempst P., Hartl F. U. Function in protein folding of TRiC, a cytosolic ring complex containing TCP-1 and structurally related subunits. EMBO J. 1992 Dec;11(13):4767–4778. doi: 10.1002/j.1460-2075.1992.tb05582.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gao Y., Thomas J. O., Chow R. L., Lee G. H., Cowan N. J. A cytoplasmic chaperonin that catalyzes beta-actin folding. Cell. 1992 Jun 12;69(6):1043–1050. doi: 10.1016/0092-8674(92)90622-j. [DOI] [PubMed] [Google Scholar]
  15. Geissler S., Pereira G., Spang A., Knop M., Souès S., Kilmartin J., Schiebel E. The spindle pole body component Spc98p interacts with the gamma-tubulin-like Tub4p of Saccharomyces cerevisiae at the sites of microtubule attachment. EMBO J. 1996 Aug 1;15(15):3899–3911. [PMC free article] [PubMed] [Google Scholar]
  16. Horio T., Uzawa S., Jung M. K., Oakley B. R., Tanaka K., Yanagida M. The fission yeast gamma-tubulin is essential for mitosis and is localized at microtubule organizing centers. J Cell Sci. 1991 Aug;99(Pt 4):693–700. doi: 10.1242/jcs.99.4.693. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Huffaker T. C., Hoyt M. A., Botstein D. Genetic analysis of the yeast cytoskeleton. Annu Rev Genet. 1987;21:259–284. doi: 10.1146/annurev.ge.21.120187.001355. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Hutter K. J., Eipel H. E. Microbial determinations by flow cytometry. J Gen Microbiol. 1979 Aug;113(2):369–375. doi: 10.1099/00221287-113-2-369. [DOI] [PubMed] [Google Scholar]
  21. Katz W., Weinstein B., Solomon F. Regulation of tubulin levels and microtubule assembly in Saccharomyces cerevisiae: consequences of altered tubulin gene copy number. Mol Cell Biol. 1990 Oct;10(10):5286–5294. doi: 10.1128/mcb.10.10.5286. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Knop M., Finger A., Braun T., Hellmuth K., Wolf D. H. Der1, a novel protein specifically required for endoplasmic reticulum degradation in yeast. EMBO J. 1996 Feb 15;15(4):753–763. [PMC free article] [PubMed] [Google Scholar]
  23. Knop M., Pereira G., Geissler S., Grein K., Schiebel E. The spindle pole body component Spc97p interacts with the gamma-tubulin of Saccharomyces cerevisiae and functions in microtubule organization and spindle pole body duplication. EMBO J. 1997 Apr 1;16(7):1550–1564. doi: 10.1093/emboj/16.7.1550. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Knop M., Schiebel E. Spc98p and Spc97p of the yeast gamma-tubulin complex mediate binding to the spindle pole body via their interaction with Spc110p. EMBO J. 1997 Dec 1;16(23):6985–6995. doi: 10.1093/emboj/16.23.6985. [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. Kranz J. E., Holm C. Cloning by function: an alternative approach for identifying yeast homologs of genes from other organisms. Proc Natl Acad Sci U S A. 1990 Sep;87(17):6629–6633. doi: 10.1073/pnas.87.17.6629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kubota H., Hynes G., Willison K. The chaperonin containing t-complex polypeptide 1 (TCP-1). Multisubunit machinery assisting in protein folding and assembly in the eukaryotic cytosol. Eur J Biochem. 1995 May 15;230(1):3–16. doi: 10.1111/j.1432-1033.1995.tb20527.x. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. 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]
  30. Li W. Z., Lin P., Frydman J., Boal T. R., Cardillo T. S., Richard L. M., Toth D., Lichtman M. A., Hartl F. U., Sherman F. Tcp20, a subunit of the eukaryotic TRiC chaperonin from humans and yeast. J Biol Chem. 1994 Jul 15;269(28):18616–18622. [PubMed] [Google Scholar]
  31. Lupas A. Coiled coils: new structures and new functions. Trends Biochem Sci. 1996 Oct;21(10):375–382. [PubMed] [Google Scholar]
  32. Marschall L. G., Jeng R. L., Mulholland J., Stearns T. Analysis of Tub4p, a yeast gamma-tubulin-like protein: implications for microtubule-organizing center function. J Cell Biol. 1996 Jul;134(2):443–454. doi: 10.1083/jcb.134.2.443. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Melki R., Vainberg I. E., Chow R. L., Cowan N. J. Chaperonin-mediated folding of vertebrate actin-related protein and gamma-tubulin. J Cell Biol. 1993 Sep;122(6):1301–1310. doi: 10.1083/jcb.122.6.1301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Miklos D., Caplan S., Mertens D., Hynes G., Pitluk Z., Kashi Y., Harrison-Lavoie K., Stevenson S., Brown C., Barrell B. Primary structure and function of a second essential member of the heterooligomeric TCP1 chaperonin complex of yeast, TCP1 beta. Proc Natl Acad Sci U S A. 1994 Mar 29;91(7):2743–2747. doi: 10.1073/pnas.91.7.2743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. Moudjou M., Bordes N., Paintrand M., Bornens M. gamma-Tubulin in mammalian cells: the centrosomal and the cytosolic forms. J Cell Sci. 1996 Apr;109(Pt 4):875–887. doi: 10.1242/jcs.109.4.875. [DOI] [PubMed] [Google Scholar]
  37. Mumberg D., Müller R., Funk M. Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. Gene. 1995 Apr 14;156(1):119–122. doi: 10.1016/0378-1119(95)00037-7. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Nitsch M., Klumpp M., Lupas A., Baumeister W. The thermosome: alternating alpha and beta-subunits within the chaperonin of the archaeon Thermoplasma acidophilum. J Mol Biol. 1997 Mar 21;267(1):142–149. doi: 10.1006/jmbi.1996.0849. [DOI] [PubMed] [Google Scholar]
  40. Oakley B. R., Oakley C. E., Yoon Y., Jung M. K. Gamma-tubulin is a component of the spindle pole body that is essential for microtubule function in Aspergillus nidulans. Cell. 1990 Jun 29;61(7):1289–1301. doi: 10.1016/0092-8674(90)90693-9. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. 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]
  43. 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]
  44. 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]
  45. Schiestl R. H., Gietz R. D. High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr Genet. 1989 Dec;16(5-6):339–346. doi: 10.1007/BF00340712. [DOI] [PubMed] [Google Scholar]
  46. Schuler G. D., Altschul S. F., Lipman D. J. A workbench for multiple alignment construction and analysis. Proteins. 1991;9(3):180–190. doi: 10.1002/prot.340090304. [DOI] [PubMed] [Google Scholar]
  47. Shang H. S., Wong S. M., Tan H. M., Wu M. YKE2, a yeast nuclear gene encoding a protein showing homology to mouse KE2 and containing a putative leucine-zipper motif. Gene. 1994 Dec 30;151(1-2):197–201. doi: 10.1016/0378-1119(94)90656-4. [DOI] [PubMed] [Google Scholar]
  48. 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]
  49. Silver L. M., Artzt K., Bennett D. A major testicular cell protein specified by a mouse T/t complex gene. Cell. 1979 Jun;17(2):275–284. doi: 10.1016/0092-8674(79)90153-3. [DOI] [PubMed] [Google Scholar]
  50. Silver L. M., Kleene K. C., Distel R. J., Hecht N. B. Synthesis of mouse t complex proteins during haploid stages of spermatogenesis. Dev Biol. 1987 Feb;119(2):605–608. doi: 10.1016/0012-1606(87)90063-7. [DOI] [PubMed] [Google Scholar]
  51. Sobel S. G., Snyder M. A highly divergent gamma-tubulin gene is essential for cell growth and proper microtubule organization in Saccharomyces cerevisiae. J Cell Biol. 1995 Dec;131(6 Pt 2):1775–1788. doi: 10.1083/jcb.131.6.1775. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Solomon F. Analyses of the cytoskeleton in Saccharomyces cerevisiae. Annu Rev Cell Biol. 1991;7:633–662. doi: 10.1146/annurev.cb.07.110191.003221. [DOI] [PubMed] [Google Scholar]
  53. 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]
  54. Spang A., Geissler S., Grein K., Schiebel E. gamma-Tubulin-like Tub4p of Saccharomyces cerevisiae is associated with the spindle pole body substructures that organize microtubules and is required for mitotic spindle formation. J Cell Biol. 1996 Jul;134(2):429–441. doi: 10.1083/jcb.134.2.429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Stearns T., Evans L., Kirschner M. Gamma-tubulin is a highly conserved component of the centrosome. Cell. 1991 May 31;65(5):825–836. doi: 10.1016/0092-8674(91)90390-k. [DOI] [PubMed] [Google Scholar]
  56. 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]
  57. Stearns T., Kirschner M. In vitro reconstitution of centrosome assembly and function: the central role of gamma-tubulin. Cell. 1994 Feb 25;76(4):623–637. doi: 10.1016/0092-8674(94)90503-7. [DOI] [PubMed] [Google Scholar]
  58. Sternlicht H., Farr G. W., Sternlicht M. L., Driscoll J. K., Willison K., Yaffe M. B. The t-complex polypeptide 1 complex is a chaperonin for tubulin and actin in vivo. Proc Natl Acad Sci U S A. 1993 Oct 15;90(20):9422–9426. doi: 10.1073/pnas.90.20.9422. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Tian G., Huang Y., Rommelaere H., Vandekerckhove J., Ampe C., Cowan N. J. Pathway leading to correctly folded beta-tubulin. Cell. 1996 Jul 26;86(2):287–296. doi: 10.1016/s0092-8674(00)80100-2. [DOI] [PubMed] [Google Scholar]
  60. Tian G., Lewis S. A., Feierbach B., Stearns T., Rommelaere H., Ampe C., Cowan N. J. Tubulin subunits exist in an activated conformational state generated and maintained by protein cofactors. J Cell Biol. 1997 Aug 25;138(4):821–832. doi: 10.1083/jcb.138.4.821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Tsuchiya H., Iseda T., Hino O. Identification of a novel protein (VBP-1) binding to the von Hippel-Lindau (VHL) tumor suppressor gene product. Cancer Res. 1996 Jul 1;56(13):2881–2885. [PubMed] [Google Scholar]
  62. Ursic D., Culbertson M. R. The yeast homolog to mouse Tcp-1 affects microtubule-mediated processes. Mol Cell Biol. 1991 May;11(5):2629–2640. doi: 10.1128/mcb.11.5.2629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Vinh D. B., Drubin D. G. A yeast TCP-1-like protein is required for actin function in vivo. Proc Natl Acad Sci U S A. 1994 Sep 13;91(19):9116–9120. doi: 10.1073/pnas.91.19.9116. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Wach A., Brachat A., Alberti-Segui C., Rebischung C., Philippsen P. Heterologous HIS3 marker and GFP reporter modules for PCR-targeting in Saccharomyces cerevisiae. Yeast. 1997 Sep 15;13(11):1065–1075. doi: 10.1002/(SICI)1097-0061(19970915)13:11<1065::AID-YEA159>3.0.CO;2-K. [DOI] [PubMed] [Google Scholar]
  65. Wach A., Brachat A., Pöhlmann R., Philippsen P. New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast. 1994 Dec;10(13):1793–1808. doi: 10.1002/yea.320101310. [DOI] [PubMed] [Google Scholar]
  66. Waldmann T., Lupas A., Kellermann J., Peters J., Baumeister W. Primary structure of the thermosome from Thermoplasma acidophilum. Biol Chem Hoppe Seyler. 1995 Feb;376(2):119–126. doi: 10.1515/bchm3.1995.376.2.119. [DOI] [PubMed] [Google Scholar]
  67. Yaffe M. B., Farr G. W., Miklos D., Horwich A. L., Sternlicht M. L., Sternlicht H. TCP1 complex is a molecular chaperone in tubulin biogenesis. Nature. 1992 Jul 16;358(6383):245–248. doi: 10.1038/358245a0. [DOI] [PubMed] [Google Scholar]
  68. Young M. R., Tye B. K. Mcm2 and Mcm3 are constitutive nuclear proteins that exhibit distinct isoforms and bind chromatin during specific cell cycle stages of Saccharomyces cerevisiae. Mol Biol Cell. 1997 Aug;8(8):1587–1601. doi: 10.1091/mbc.8.8.1587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Zheng Y., Wong M. L., Alberts B., Mitchison T. Nucleation of microtubule assembly by a gamma-tubulin-containing ring complex. Nature. 1995 Dec 7;378(6557):578–583. doi: 10.1038/378578a0. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES