Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1994 Jul 1;301(Pt 1):105–110. doi: 10.1042/bj3010105

Role of 300 kDa complexes as intermediates in tubulin folding and dimerization: characterization of a 25 kDa cytosolic protein involved in the GTP-dependent release of monomeric tubulin.

R Paciucci 1
PMCID: PMC1137149  PMID: 8037656

Abstract

beta-Tubulin synthesized in vitro in rabbit reticulocyte lysate is found associated with 900 kDa complexes (C900) containing T Complex Polypeptide 1 (TCP1), heat-shock protein (hsp) 70 and other unidentified proteins, with smaller 300 kDa complexes (C300) of unknown nature, in dimeric association with reticulocyte alpha-tubulin and in monomeric forms. Pulse-chase experiments indicated that production of fully functional beta-tubulin was preceded by its association with C900 and C300 multimolecular complexes and by the appearance of beta-monomers. The high-molecular-mass forms appeared as intermediate products in the process leading to fully functional dimerizable beta-tubulin. C300-associated tubulin can be released as beta-monomer by addition of a cofactor present in reticulocyte lysate. Here a 25 kDa protein which releases tubulin monomers from C300 has been identified and characterized. The protein specifically released monomers from C300, but not from C900, in a process favoured by GTP.

Full text

PDF
107

Images in this article

106Figure 1
on p.106
107Figure 2
on p.107
108Figure 3
on p.108
108Figure 5
on p.108

Selected References

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

  1. Bischoff F. R., Ponstingl H. Mitotic regulator protein RCC1 is complexed with a nuclear ras-related polypeptide. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10830–10834. doi: 10.1073/pnas.88.23.10830. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  3. Ellis R. J., van der Vies S. M. Molecular chaperones. Annu Rev Biochem. 1991;60:321–347. doi: 10.1146/annurev.bi.60.070191.001541. [DOI] [PubMed] [Google Scholar]
  4. Farr G. W., Yaffe M. B., Sternlicht H. Alpha-tubulin influences nucleotide binding to beta-tubulin: an assay using picomoles of unpurified protein. Proc Natl Acad Sci U S A. 1990 Jul;87(13):5041–5045. doi: 10.1073/pnas.87.13.5041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Fontalba A., Paciucci R., Avila J., Zabala J. C. Incorporation of tubulin subunits into dimers requires GTP hydrolysis. J Cell Sci. 1993 Oct;106(Pt 2):627–632. doi: 10.1242/jcs.106.2.627. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. Gao Y., Vainberg I. E., Chow R. L., Cowan N. J. Two cofactors and cytoplasmic chaperonin are required for the folding of alpha- and beta-tubulin. Mol Cell Biol. 1993 Apr;13(4):2478–2485. doi: 10.1128/mcb.13.4.2478. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Goloubinoff P., Christeller J. T., Gatenby A. A., Lorimer G. H. Reconstitution of active dimeric ribulose bisphosphate carboxylase from an unfoleded state depends on two chaperonin proteins and Mg-ATP. Nature. 1989 Dec 21;342(6252):884–889. doi: 10.1038/342884a0. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Landry S. J., Gierasch L. M. Recognition of nascent polypeptides for targeting and folding. Trends Biochem Sci. 1991 Apr;16(4):159–163. doi: 10.1016/0968-0004(91)90060-9. [DOI] [PubMed] [Google Scholar]
  12. Langer T., Lu C., Echols H., Flanagan J., Hayer M. K., Hartl F. U. Successive action of DnaK, DnaJ and GroEL along the pathway of chaperone-mediated protein folding. Nature. 1992 Apr 23;356(6371):683–689. doi: 10.1038/356683a0. [DOI] [PubMed] [Google Scholar]
  13. Lewis V. A., Hynes G. M., Zheng D., Saibil H., Willison K. T-complex polypeptide-1 is a subunit of a heteromeric particle in the eukaryotic cytosol. Nature. 1992 Jul 16;358(6383):249–252. doi: 10.1038/358249a0. [DOI] [PubMed] [Google Scholar]
  14. Manning-Krieg U. C., Scherer P. E., Schatz G. Sequential action of mitochondrial chaperones in protein import into the matrix. EMBO J. 1991 Nov;10(11):3273–3280. doi: 10.1002/j.1460-2075.1991.tb04891.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Marchesi V. T., Ngo N. In vitro assembly of multiprotein complexes containing alpha, beta, and gamma tubulin, heat shock protein HSP70, and elongation factor 1 alpha. Proc Natl Acad Sci U S A. 1993 Apr 1;90(7):3028–3032. doi: 10.1073/pnas.90.7.3028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Martin J., Langer T., Boteva R., Schramel A., Horwich A. L., Hartl F. U. Chaperonin-mediated protein folding at the surface of groEL through a 'molten globule'-like intermediate. Nature. 1991 Jul 4;352(6330):36–42. doi: 10.1038/352036a0. [DOI] [PubMed] [Google Scholar]
  17. Ruddon R. W., Krzesicki R. F., Norton S. E., Beebe J. S., Peters B. P., Perini F. Detection of a glycosylated, incompletely folded form of chorionic gonadotropin beta subunit that is a precursor of hormone assembly in trophoblastic cells. J Biol Chem. 1987 Sep 15;262(26):12533–12540. [PubMed] [Google Scholar]
  18. 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]
  19. Strickland T. W., Pierce J. G. The beta subunits of glycoprotein hormones. Formation of three-dimensional structure during cell-free biosynthesis of lutropin-beta. J Biol Chem. 1985 May 10;260(9):5816–5819. [PubMed] [Google Scholar]
  20. Strickland T. W., Puett D. The kinetic and equilibrium parameters of subunit association and gonadotropin dissociation. J Biol Chem. 1982 Mar 25;257(6):2954–2960. [PubMed] [Google Scholar]
  21. 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]
  22. Yaffe M. B., Farr G. W., Sternlicht H. Kinetics of beta-tubulin exchange following translation. Evidence for a slow conformational change in beta-tubulin necessary for incorporation into heterodimers. J Biol Chem. 1989 Nov 15;264(32):19045–19051. [PubMed] [Google Scholar]
  23. Yaffe M. B., Farr G. W., Sternlicht H. Translation of beta-tubulin mRNA in vitro generates multiple molecular forms. J Biol Chem. 1988 Nov 5;263(31):16023–16031. [PubMed] [Google Scholar]
  24. Yen T. J., Machlin P. S., Cleveland D. W. Autoregulated instability of beta-tubulin mRNAs by recognition of the nascent amino terminus of beta-tubulin. Nature. 1988 Aug 18;334(6183):580–585. doi: 10.1038/334580a0. [DOI] [PubMed] [Google Scholar]
  25. Zabala J. C., Cowan N. J. Tubulin dimer formation via the release of alpha- and beta-tubulin monomers from multimolecular complexes. Cell Motil Cytoskeleton. 1992;23(3):222–230. doi: 10.1002/cm.970230306. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES