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. 1994 May;14(5):2895–2904. doi: 10.1128/mcb.14.5.2895

Facilitated folding of actins and tubulins occurs via a nucleotide-dependent interaction between cytoplasmic chaperonin and distinctive folding intermediates.

R Melki 1, N J Cowan 1
PMCID: PMC358657  PMID: 7909354

Abstract

In the cytoplasm of eukaryotes, the folding of actins and tubulins is facilitated via interaction with a heteromeric toroidal complex (cytoplasmic chaperonin). The folding reaction consists of the formation of a binary complex between the unfolded target protein and the chaperonin, followed by the ultimate release of the native polypeptide in an ATP-dependent reaction. Here we show that the mitochondrial chaperonin (cpn60) and the cytoplasmic chaperonin both recognize a range of target proteins with different relative affinities; however, the cytoplasmic chaperonin shows the highest affinity for intermediates derived from unfolded tubulins and actins. These high-affinity actin and tubulin folding intermediates are distinct from the "molten globule" intermediates formed by noncytoskeletal target proteins in that they form relatively slowly. We show that the interaction between cytoplasmic chaperonin and unfolded target proteins depends on the chaperonin being in its ADP-bound state and that the release of the target protein occurs after a transition of the chaperonin to the ATP-bound state. Our data suggest a model in which ATP hydrolysis acts as a switch between conformational forms of the cytoplasmic chaperonin that interact either strongly or weakly with unfolded substrates.

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Selected References

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

  1. Ahmad S., Gupta R. S. Cloning of a Chinese hamster protein homologous to the mouse t-complex protein TCP-1: structural similarity to the ubiquitous 'chaperonin' family of heat-shock proteins. Biochim Biophys Acta. 1990 Oct 23;1087(2):253–255. doi: 10.1016/0167-4781(90)90214-m. [DOI] [PubMed] [Google Scholar]
  2. Anfinsen C. B. Principles that govern the folding of protein chains. Science. 1973 Jul 20;181(4096):223–230. doi: 10.1126/science.181.4096.223. [DOI] [PubMed] [Google Scholar]
  3. Badcoe I. G., Smith C. J., Wood S., Halsall D. J., Holbrook J. J., Lund P., Clarke A. R. Binding of a chaperonin to the folding intermediates of lactate dehydrogenase. Biochemistry. 1991 Sep 24;30(38):9195–9200. doi: 10.1021/bi00102a010. [DOI] [PubMed] [Google Scholar]
  4. Bigay J., Deterre P., Pfister C., Chabre M. Fluoride complexes of aluminium or beryllium act on G-proteins as reversibly bound analogues of the gamma phosphate of GTP. EMBO J. 1987 Oct;6(10):2907–2913. doi: 10.1002/j.1460-2075.1987.tb02594.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bigay J., Deterre P., Pfister C., Chabre M. Fluoroaluminates activate transducin-GDP by mimicking the gamma-phosphate of GTP in its binding site. FEBS Lett. 1985 Oct 28;191(2):181–185. doi: 10.1016/0014-5793(85)80004-1. [DOI] [PubMed] [Google Scholar]
  6. Bochkareva E. S., Lissin N. M., Flynn G. C., Rothman J. E., Girshovich A. S. Positive cooperativity in the functioning of molecular chaperone GroEL. J Biol Chem. 1992 Apr 5;267(10):6796–6800. [PubMed] [Google Scholar]
  7. Bochkareva E. S., Lissin N. M., Girshovich A. S. Transient association of newly synthesized unfolded proteins with the heat-shock GroEL protein. Nature. 1988 Nov 17;336(6196):254–257. doi: 10.1038/336254a0. [DOI] [PubMed] [Google Scholar]
  8. Bourne H. R., Sanders D. A., McCormick F. The GTPase superfamily: conserved structure and molecular mechanism. Nature. 1991 Jan 10;349(6305):117–127. doi: 10.1038/349117a0. [DOI] [PubMed] [Google Scholar]
  9. Carlier M. F., Didry D., Melki R., Chabre M., Pantaloni D. Stabilization of microtubules by inorganic phosphate and its structural analogues, the fluoride complexes of aluminum and beryllium. Biochemistry. 1988 May 17;27(10):3555–3559. doi: 10.1021/bi00410a005. [DOI] [PubMed] [Google Scholar]
  10. Cheng M. Y., Hartl F. U., Martin J., Pollock R. A., Kalousek F., Neupert W., Hallberg E. M., Hallberg R. L., Horwich A. L. Mitochondrial heat-shock protein hsp60 is essential for assembly of proteins imported into yeast mitochondria. Nature. 1989 Feb 16;337(6208):620–625. doi: 10.1038/337620a0. [DOI] [PubMed] [Google Scholar]
  11. Cleveland D. W., Lopata M. A., MacDonald R. J., Cowan N. J., Rutter W. J., Kirschner M. W. Number and evolutionary conservation of alpha- and beta-tubulin and cytoplasmic beta- and gamma-actin genes using specific cloned cDNA probes. Cell. 1980 May;20(1):95–105. doi: 10.1016/0092-8674(80)90238-x. [DOI] [PubMed] [Google Scholar]
  12. Combeau C., Carlier M. F. Characterization of the aluminum and beryllium fluoride species bound to F-actin and microtubules at the site of the gamma-phosphate of the nucleotide. J Biol Chem. 1989 Nov 15;264(32):19017–19021. [PubMed] [Google Scholar]
  13. Eisenberg E., Hill T. L. Muscle contraction and free energy transduction in biological systems. Science. 1985 Mar 1;227(4690):999–1006. doi: 10.1126/science.3156404. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. 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]
  17. 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]
  18. Gething M. J., Sambrook J. Protein folding in the cell. Nature. 1992 Jan 2;355(6355):33–45. doi: 10.1038/355033a0. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Gray T. E., Fersht A. R. Cooperativity in ATP hydrolysis by GroEL is increased by GroES. FEBS Lett. 1991 Nov 4;292(1-2):254–258. doi: 10.1016/0014-5793(91)80878-7. [DOI] [PubMed] [Google Scholar]
  21. Holmes K. C., Popp D., Gebhard W., Kabsch W. Atomic model of the actin filament. Nature. 1990 Sep 6;347(6288):44–49. doi: 10.1038/347044a0. [DOI] [PubMed] [Google Scholar]
  22. Horwich A. L., Low K. B., Fenton W. A., Hirshfield I. N., Furtak K. Folding in vivo of bacterial cytoplasmic proteins: role of GroEL. Cell. 1993 Sep 10;74(5):909–917. doi: 10.1016/0092-8674(93)90470-b. [DOI] [PubMed] [Google Scholar]
  23. Jackson G. S., Staniforth R. A., Halsall D. J., Atkinson T., Holbrook J. J., Clarke A. R., Burston S. G. Binding and hydrolysis of nucleotides in the chaperonin catalytic cycle: implications for the mechanism of assisted protein folding. Biochemistry. 1993 Mar 16;32(10):2554–2563. doi: 10.1021/bi00061a013. [DOI] [PubMed] [Google Scholar]
  24. Jaenicke R. Protein folding: local structures, domains, subunits, and assemblies. Biochemistry. 1991 Apr 2;30(13):3147–3161. doi: 10.1021/bi00227a001. [DOI] [PubMed] [Google Scholar]
  25. Land H., Parada L. F., Weinberg R. A. Tumorigenic conversion of primary embryo fibroblasts requires at least two cooperating oncogenes. Nature. 1983 Aug 18;304(5927):596–602. doi: 10.1038/304596a0. [DOI] [PubMed] [Google Scholar]
  26. Lees-Miller J. P., Helfman D. M., Schroer T. A. A vertebrate actin-related protein is a component of a multisubunit complex involved in microtubule-based vesicle motility. Nature. 1992 Sep 17;359(6392):244–246. doi: 10.1038/359244a0. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. Melki R., Carlier M. F., Pantaloni D. Direct evidence for GTP and GDP-Pi intermediates in microtubule assembly. Biochemistry. 1990 Sep 25;29(38):8921–8932. doi: 10.1021/bi00490a007. [DOI] [PubMed] [Google Scholar]
  30. Melki R., Carlier M. F. Thermodynamics of tubulin polymerization into zinc sheets: assembly is not regulated by GTP hydrolysis. Biochemistry. 1993 Apr 6;32(13):3405–3413. doi: 10.1021/bi00064a026. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Mizobata T., Akiyama Y., Ito K., Yumoto N., Kawata Y. Effects of the chaperonin GroE on the refolding of tryptophanase from Escherichia coli. Refolding is enhanced in the presence of ADP. J Biol Chem. 1992 Sep 5;267(25):17773–17779. [PubMed] [Google Scholar]
  33. Moreau P. L., Carlier M. F. RecA protein-promoted cleavage of LexA repressor in the presence of ADP and structural analogues of inorganic phosphate, the fluoride complexes of aluminum and beryllium. J Biol Chem. 1989 Feb 5;264(4):2302–2306. [PubMed] [Google Scholar]
  34. Ostermann J., Horwich A. L., Neupert W., Hartl F. U. Protein folding in mitochondria requires complex formation with hsp60 and ATP hydrolysis. Nature. 1989 Sep 14;341(6238):125–130. doi: 10.1038/341125a0. [DOI] [PubMed] [Google Scholar]
  35. Pines J., Hunter T. Isolation of a human cyclin cDNA: evidence for cyclin mRNA and protein regulation in the cell cycle and for interaction with p34cdc2. Cell. 1989 Sep 8;58(5):833–846. doi: 10.1016/0092-8674(89)90936-7. [DOI] [PubMed] [Google Scholar]
  36. Prendergast G. C., Lawe D., Ziff E. B. Association of Myn, the murine homolog of max, with c-Myc stimulates methylation-sensitive DNA binding and ras cotransformation. Cell. 1991 May 3;65(3):395–407. doi: 10.1016/0092-8674(91)90457-a. [DOI] [PubMed] [Google Scholar]
  37. Rommelaere H., Van Troys M., Gao Y., Melki R., Cowan N. J., Vandekerckhove J., Ampe C. Eukaryotic cytosolic chaperonin contains t-complex polypeptide 1 and seven related subunits. Proc Natl Acad Sci U S A. 1993 Dec 15;90(24):11975–11979. doi: 10.1073/pnas.90.24.11975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Rothman J. E. Polypeptide chain binding proteins: catalysts of protein folding and related processes in cells. Cell. 1989 Nov 17;59(4):591–601. doi: 10.1016/0092-8674(89)90005-6. [DOI] [PubMed] [Google Scholar]
  39. Rychlik W., Domier L. L., Gardner P. R., Hellmann G. M., Rhoads R. E. Amino acid sequence of the mRNA cap-binding protein from human tissues. Proc Natl Acad Sci U S A. 1987 Feb;84(4):945–949. doi: 10.1073/pnas.84.4.945. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Saibil H. R., Zheng D., Roseman A. M., Hunter A. S., Watson G. M., Chen S., Auf Der Mauer A., O'Hara B. P., Wood S. P., Mann N. H. ATP induces large quaternary rearrangements in a cage-like chaperonin structure. Curr Biol. 1993 May 1;3(5):265–273. doi: 10.1016/0960-9822(93)90176-o. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  43. Todd M. J., Viitanen P. V., Lorimer G. H. Hydrolysis of adenosine 5'-triphosphate by Escherichia coli GroEL: effects of GroES and potassium ion. Biochemistry. 1993 Aug 24;32(33):8560–8567. doi: 10.1021/bi00084a024. [DOI] [PubMed] [Google Scholar]
  44. Viitanen P. V., Gatenby A. A., Lorimer G. H. Purified chaperonin 60 (groEL) interacts with the nonnative states of a multitude of Escherichia coli proteins. Protein Sci. 1992 Mar;1(3):363–369. doi: 10.1002/pro.5560010308. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Viitanen P. V., Lorimer G. H., Seetharam R., Gupta R. S., Oppenheim J., Thomas J. O., Cowan N. J. Mammalian mitochondrial chaperonin 60 functions as a single toroidal ring. J Biol Chem. 1992 Jan 15;267(2):695–698. [PubMed] [Google Scholar]
  46. Villasante A., Wang D., Dobner P., Dolph P., Lewis S. A., Cowan N. J. Six mouse alpha-tubulin mRNAs encode five distinct isotypes: testis-specific expression of two sister genes. Mol Cell Biol. 1986 Jul;6(7):2409–2419. doi: 10.1128/mcb.6.7.2409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wang D., Villasante A., Lewis S. A., Cowan N. J. The mammalian beta-tubulin repertoire: hematopoietic expression of a novel, heterologous beta-tubulin isotype. J Cell Biol. 1986 Nov;103(5):1903–1910. doi: 10.1083/jcb.103.5.1903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. 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]
  49. Zheng Y., Jung M. K., Oakley B. R. Gamma-tubulin is present in Drosophila melanogaster and Homo sapiens and is associated with the centrosome. Cell. 1991 May 31;65(5):817–823. doi: 10.1016/0092-8674(91)90389-g. [DOI] [PubMed] [Google Scholar]

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