Home LiteratureArticle Details
PMID: 11950928 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

Reconstitution and characterization of budding yeast gamma-tubulin complex.

Molecular biology of the cell ·Vol. 13 ·No. 4 ·2002-04-00 ·Pages 1144-57

Vinh DB, Kern JW, Hancock WO, Howard J, Davis TN

Abstract

Nucleation of microtubules is central to assembly of the mitotic spindle, which is required for each cell division. gamma-Tubulin is a universal component essential for microtubule nucleation from centrosomes. To elucidate the mechanism of microtubule nucleation in budding yeast we reconstituted and characterized the yeast gamma-tubulin complex (Tub4p complex) produced in insect cells. The recombinant complex has the same sedimentation coefficient (11.6 S) as the native complex in yeast cell extracts and contains one molecule of Spc97p, one molecule of Spc98p, and two molecules of Tub4p. The reconstituted Tub4p complex binds preformed microtubules and has a low nucleating activity, allowing us to begin a detailed analysis of conditions that enhance this nucleating activity. We tested whether binding of the recombinant Tub4p complex to the spindle pole body docking protein Spc110p affects its nucleating activity. The solubility of recombinant Spc110p in insect cells is improved by coexpression with yeast calmodulin (Cmd1p). The Spc110p/Cmd1p complex has a small sedimentation coefficient (4.2 S) and a large Stokes radius (14.3 nm), indicative of an elongated structure. The Tub4p complex binds Spc110p/Cmd1p via Spc98p and the K(d) for binding is 150 nM. The low nucleation activity of the Tub4p complex is not enhanced when it is bound to Spc110p/Cmd1p, suggesting that it requires additional components or modifications to achieve robust activity. Finally, we report the identification of a large 22 S Tub4p complex in yeast extract that contains multimers of Spc97p similar to gamma-tubulin ring complexes found in higher eukaryotic cells.

MeSH Terms
Animals Baculoviridae/genetics Blotting, Western Cell Line Cell Nucleus/metabolism Centrifugation, Density Gradient Chromatography, Gel Dose-Response Relationship, Drug Electrophoresis, Polyacrylamide Gel Fungal Proteins/metabolism Glutathione Transferase/metabolism Insecta Kinetics Microtubule-Associated Proteins/metabolism Microtubules/metabolism Precipitin Tests Protein Binding Recombinant Proteins/metabolism Saccharomyces cerevisiae Proteins Saccharomycetales/metabolism Tubulin/chemistry,metabolism
Chemicals
Fungal Proteins Microtubule-Associated Proteins Recombinant Proteins SPC97 protein, S cerevisiae Saccharomyces cerevisiae Proteins TUB4 protein, S cerevisiae TUBGCP2 protein, human Tubulin Glutathione Transferase
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Vinh Dani B N
Departments of Biochemistry, University of Washington, Seattle, WA 98195, USA.
Kern Joshua W
Hancock William O
Howard Jonathon
Davis Trisha N
References (51)
51 references, click to expand
  1. Structure of the gamma-tubulin ring complex: a template for microtubule nucleation.
    Nat Cell Biol. 2000 Jun;2(6):365-70 PMID: 10854328
  2. Spc29p is a component of the Spc110p subcomplex and is essential for spindle pole body duplication.
    Proc Natl Acad Sci U S A. 1999 May 25;96(11):6205-10 PMID: 10339566
  3. Monomeric gamma -tubulin nucleates microtubules.
    J Biol Chem. 2000 Jul 21;275(29):21975-80 PMID: 10764751
  4. Characterization and reconstitution of Drosophila gamma-tubulin ring complex subunits.
    J Cell Biol. 2000 Dec 25;151(7):1513-24 PMID: 11134079
  5. The role of Xgrip210 in gamma-tubulin ring complex assembly and centrosome recruitment.
    J Cell Biol. 2000 Dec 25;151(7):1525-36 PMID: 11134080
  6. GCP5 and GCP6: two new members of the human gamma-tubulin complex.
    Mol Biol Cell. 2001 Nov;12(11):3340-52 PMID: 11694571
  7. Determination of molecular weights and frictional ratios of proteins in impure systems by use of gel filtration and density gradient centrifugation. Application to crude preparations of sulfite and hydroxylamine reductases.
    Biochim Biophys Acta. 1966 Feb 7;112(2):346-62 PMID: 5329026
  8. The role of spindle pole bodies and modified microtubule ends in the initiation of microtubule assembly in Saccharomyces cerevisiae.
    J Cell Sci. 1978 Apr;30:331-52 PMID: 348712
  9. Microtubule assembly nucleated by isolated centrosomes.
    Nature. 1984 Nov 15-21;312(5991):232-7 PMID: 6504137
  10. 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):1289-301 PMID: 2194669
  11. Components of the yeast spindle and spindle pole body.
    J Cell Biol. 1990 Nov;111(5 Pt 1):1913-27 PMID: 2229181
  12. Preparation of modified tubulins.
    Methods Enzymol. 1991;196:478-85 PMID: 2034137
  13. Gamma-tubulin is present in Drosophila melanogaster and Homo sapiens and is associated with the centrosome.
    Cell. 1991 May 31;65(5):817-23 PMID: 1904010
  14. Gamma-tubulin is a highly conserved component of the centrosome.
    Cell. 1991 May 31;65(5):825-36 PMID: 1840506
  15. Can calmodulin function without binding calcium?
    Cell. 1991 Jun 14;65(6):949-59 PMID: 2044154
  16. 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 PMID: 1770000
  17. Gamma-tubulin is a centrosomal protein required for cell cycle-dependent microtubule nucleation.
    Nature. 1992 Mar 5;356(6364):80-3 PMID: 1538786
  18. A spacer protein in the Saccharomyces cerevisiae spindle poly body whose transcript is cell cycle-regulated.
    J Cell Biol. 1993 Dec;123(5):1175-84 PMID: 7503995
  19. The essential mitotic target of calmodulin is the 110-kilodalton component of the spindle pole body in Saccharomyces cerevisiae.
    Mol Cell Biol. 1993 Dec;13(12):7913-24 PMID: 8247006
  20. Centrosome assembly in vitro: role of gamma-tubulin recruitment in Xenopus sperm aster formation.
    J Cell Biol. 1994 Jan;124(1-2):19-31 PMID: 8294501
  21. In vitro reconstitution of centrosome assembly and function: the central role of gamma-tubulin.
    Cell. 1994 Feb 25;76(4):623-37 PMID: 8124706
  22. gamma-tubulin is a minus end-specific microtubule binding protein.
    J Cell Biol. 1995 Oct;131(1):207-14 PMID: 7559777
  23. Nucleation of microtubule assembly by a gamma-tubulin-containing ring complex.
    Nature. 1995 Dec 7;378(6557):578-83 PMID: 8524390
  24. Microtubule nucleation by gamma-tubulin-containing rings in the centrosome.
    Nature. 1995 Dec 7;378(6557):638-40 PMID: 8524401
  25. 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):1775-88 PMID: 8557744
  26. Spc42p: a phosphorylated component of the S. cerevisiae spindle pole body (SPD) with an essential function during SPB duplication.
    J Cell Biol. 1996 Mar;132(5):887-901 PMID: 8603920
  27. Role of calmodulin and Spc110p interaction in the proper assembly of spindle pole body compenents.
    J Cell Biol. 1996 Apr;133(1):111-24 PMID: 8601600
  28. Polarity and nucleation of microtubules in polarized epithelial cells.
    Cell Motil Cytoskeleton. 1995;32(4):273-88 PMID: 8608606
  29. 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):429-41 PMID: 8707827
  30. Analysis of Tub4p, a yeast gamma-tubulin-like protein: implications for microtubule-organizing center function.
    J Cell Biol. 1996 Jul;134(2):443-54 PMID: 8707828
  31. The spacer protein Spc110p targets calmodulin to the central plaque of the yeast spindle pole body.
    J Cell Sci. 1996 Sep;109 ( Pt 9):2229-37 PMID: 8886974
  32. Spc110p: assembly properties and role in the connection of nuclear microtubules to the yeast spindle pole body.
    EMBO J. 1996 Sep 2;15(17):4592-602 PMID: 8887551
  33. Centrosomes isolated from Spisula solidissima oocytes contain rings and an unusual stoichiometric ratio of alpha/beta tubulin.
    J Cell Biol. 1997 Apr 7;137(1):193-202 PMID: 9105047
  34. 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):1550-64 PMID: 9130700
  35. Heterologous HIS3 marker and GFP reporter modules for PCR-targeting in Saccharomyces cerevisiae.
    Yeast. 1997 Sep 15;13(11):1065-75 PMID: 9290211
  36. 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):6985-95 PMID: 9384578
  37. Pericentrin and gamma-tubulin form a protein complex and are organized into a novel lattice at the centrosome.
    J Cell Biol. 1998 Apr 6;141(1):163-74 PMID: 9531556
  38. The mammalian gamma-tubulin complex contains homologues of the yeast spindle pole body components spc97p and spc98p.
    J Cell Biol. 1998 May 4;141(3):663-74 PMID: 9566967
  39. Xgrip109: a gamma tubulin-associated protein with an essential role in gamma tubulin ring complex (gammaTuRC) assembly and centrosome function.
    J Cell Biol. 1998 May 4;141(3):675-87 PMID: 9566968
  40. Characterization of the human homologue of the yeast spc98p and its association with gamma-tubulin.
    J Cell Biol. 1998 May 4;141(3):689-701 PMID: 9566969
  41. The disassembly and reassembly of functional centrosomes in vitro.
    Proc Natl Acad Sci U S A. 1998 Aug 4;95(16):9295-300 PMID: 9689074
  42. A genetic analysis of interactions with Spc110p reveals distinct functions of Spc97p and Spc98p, components of the yeast gamma-tubulin complex.
    Mol Biol Cell. 1998 Aug;9(8):2201-16 PMID: 9693376
  43. Recruitment of the gamma-tubulin ring complex to Drosophila salt-stripped centrosome scaffolds.
    J Cell Biol. 1998 Aug 10;142(3):775-86 PMID: 9700165
  44. Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe.
    Yeast. 1998 Jul;14(10):943-51 PMID: 9717240
  45. Characterization of two related Drosophila gamma-tubulin complexes that differ in their ability to nucleate microtubules.
    J Cell Biol. 1999 Feb 22;144(4):721-33 PMID: 10037793
  46. Human 76p: A new member of the gamma-tubulin-associated protein family.
    J Cell Biol. 1999 Nov 15;147(4):857-68 PMID: 10562286
  47. Identification of a human centrosomal calmodulin-binding protein that shares homology with pericentrin.
    Proc Natl Acad Sci U S A. 2000 May 23;97(11):5919-23 PMID: 10823944
  48. Immunostructural evidence for the template mechanism of microtubule nucleation.
    Nat Cell Biol. 2000 Jun;2(6):352-7 PMID: 10854326
  49. A new function for the gamma-tubulin ring complex as a microtubule minus-end cap.
    Nat Cell Biol. 2000 Jun;2(6):358-64 PMID: 10854327
  50. Localization of core spindle pole body (SPB) components during SPB duplication in Saccharomyces cerevisiae.
    J Cell Biol. 1999 May 17;145(4):809-23 PMID: 10330408
  51. Gamma-tubulin nucleation: template or protofilament?
    Nat Cell Biol. 2000 Jun;2(6):E93-6 PMID: 10854332
Article Info
Journal
Molecular biology of the cell
Abbr.
Mol Biol Cell
ISSN
1059-1524
DOI
10.1091/mbc.02-01-0607
Published
2002-04-00
Pages
1144-57
Language
English
Region
United States
NLM ID
9201390
PMCID
PMC102258
Subset
IM
Grants
NIGMS NIH HHS · R01 GM040506 · United States
NIGMS NIH HHS · F32 GM 17945 · United States
External Links
PubMed source DOI: 10.1091/mbc.02-01-0607 PMC Full Text View journal details
Analysis Services
Analysis Services

Contact

No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong

Qilu Normal University · Genelibs Bioinformatics Lab

750 Shunhua Rd, Jinan

2F, Bldg F, University Science Park

Tel: 0531-88819269

WeChat Official Account

Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.


Business Email

E-mail: product@genelibs.com

Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2