Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1999 Jan 4;18(1):75–84. doi: 10.1093/emboj/18.1.75

Compartmentation of protein folding in vivo: sequestration of non-native polypeptide by the chaperonin-GimC system.

K Siegers 1, T Waldmann 1, M R Leroux 1, K Grein 1, A Shevchenko 1, E Schiebel 1, F U Hartl 1
PMCID: PMC1171104  PMID: 9878052

Abstract

The functional coupling of protein synthesis and chaperone-assisted folding in vivo has remained largely unexplored. Here we have analysed the chaperonin-dependent folding pathway of actin in yeast. Remarkably, overexpression of a heterologous chaperonin which traps non-native polypeptides does not interfere with protein folding in the cytosol, indicating a high-level organization of folding reactions. Newly synthesized actin avoids the chaperonin trap and is effectively channelled from the ribosome to the endogenous chaperonin TRiC. Efficient actin folding on TRiC is critically dependent on the hetero-oligomeric co-chaperone GimC. By interacting with folding intermediates and with TRiC, GimC accelerates actin folding at least 5-fold and prevents the premature release of non-native protein from TRiC. We propose that TRiC and GimC form an integrated 'folding compartment' which functions in cooperation with the translation machinery. This compartment sequesters newly synthesized actin and other aggregation-sensitive polypeptides from the crowded macromolecular environment of the cytosol, thereby allowing their efficient folding.

Full Text

The Full Text of this article is available as a PDF (495.5 KB).

Selected References

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

  1. 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]
  2. Buchberger A., Schröder H., Hesterkamp T., Schönfeld H. J., Bukau B. Substrate shuttling between the DnaK and GroEL systems indicates a chaperone network promoting protein folding. J Mol Biol. 1996 Aug 23;261(3):328–333. doi: 10.1006/jmbi.1996.0465. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Ditzel L., Löwe J., Stock D., Stetter K. O., Huber H., Huber R., Steinbacher S. Crystal structure of the thermosome, the archaeal chaperonin and homolog of CCT. Cell. 1998 Apr 3;93(1):125–138. doi: 10.1016/s0092-8674(00)81152-6. [DOI] [PubMed] [Google Scholar]
  5. Eggers D. K., Welch W. J., Hansen W. J. Complexes between nascent polypeptides and their molecular chaperones in the cytosol of mammalian cells. Mol Biol Cell. 1997 Aug;8(8):1559–1573. doi: 10.1091/mbc.8.8.1559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ellis J. Proteins as molecular chaperones. 1987 Jul 30-Aug 5Nature. 328(6129):378–379. doi: 10.1038/328378a0. [DOI] [PubMed] [Google Scholar]
  7. Ewalt K. L., Hendrick J. P., Houry W. A., Hartl F. U. In vivo observation of polypeptide flux through the bacterial chaperonin system. Cell. 1997 Aug 8;90(3):491–500. doi: 10.1016/s0092-8674(00)80509-7. [DOI] [PubMed] [Google Scholar]
  8. Farr G. W., Scharl E. C., Schumacher R. J., Sondek S., Horwich A. L. Chaperonin-mediated folding in the eukaryotic cytosol proceeds through rounds of release of native and nonnative forms. Cell. 1997 Jun 13;89(6):927–937. doi: 10.1016/s0092-8674(00)80278-0. [DOI] [PubMed] [Google Scholar]
  9. Fenton W. A., Horwich A. L. GroEL-mediated protein folding. Protein Sci. 1997 Apr;6(4):743–760. doi: 10.1002/pro.5560060401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Frydman J., Hartl F. U. Principles of chaperone-assisted protein folding: differences between in vitro and in vivo mechanisms. Science. 1996 Jun 7;272(5267):1497–1502. doi: 10.1126/science.272.5267.1497. [DOI] [PubMed] [Google Scholar]
  11. Frydman J., Nimmesgern E., Ohtsuka K., Hartl F. U. Folding of nascent polypeptide chains in a high molecular mass assembly with molecular chaperones. Nature. 1994 Jul 14;370(6485):111–117. doi: 10.1038/370111a0. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Geissler S., Siegers K., Schiebel E. A novel protein complex promoting formation of functional alpha- and gamma-tubulin. EMBO J. 1998 Feb 16;17(4):952–966. doi: 10.1093/emboj/17.4.952. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Hartl F. U. Molecular chaperones in cellular protein folding. Nature. 1996 Jun 13;381(6583):571–579. doi: 10.1038/381571a0. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. James P., Pfund C., Craig E. A. Functional specificity among Hsp70 molecular chaperones. Science. 1997 Jan 17;275(5298):387–389. doi: 10.1126/science.275.5298.387. [DOI] [PubMed] [Google Scholar]
  18. Johnson J. L., Craig E. A. Protein folding in vivo: unraveling complex pathways. Cell. 1997 Jul 25;90(2):201–204. doi: 10.1016/s0092-8674(00)80327-x. [DOI] [PubMed] [Google Scholar]
  19. Kabsch W., Mannherz H. G., Suck D., Pai E. F., Holmes K. C. Atomic structure of the actin:DNase I complex. Nature. 1990 Sep 6;347(6288):37–44. doi: 10.1038/347037a0. [DOI] [PubMed] [Google Scholar]
  20. Karpova T. S., Tatchell K., Cooper J. A. Actin filaments in yeast are unstable in the absence of capping protein or fimbrin. J Cell Biol. 1995 Dec;131(6 Pt 1):1483–1493. doi: 10.1083/jcb.131.6.1483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Klumpp M., Baumeister W., Essen L. O. Structure of the substrate binding domain of the thermosome, an archaeal group II chaperonin. Cell. 1997 Oct 17;91(2):263–270. doi: 10.1016/s0092-8674(00)80408-0. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Lazarides E., Lindberg U. Actin is the naturally occurring inhibitor of deoxyribonuclease I. Proc Natl Acad Sci U S A. 1974 Dec;71(12):4742–4746. doi: 10.1073/pnas.71.12.4742. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lewis S. A., Tian G., Vainberg I. E., Cowan N. J. Chaperonin-mediated folding of actin and tubulin. J Cell Biol. 1996 Jan;132(1-2):1–4. doi: 10.1083/jcb.132.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Liou A. K., Willison K. R. Elucidation of the subunit orientation in CCT (chaperonin containing TCP1) from the subunit composition of CCT micro-complexes. EMBO J. 1997 Jul 16;16(14):4311–4316. doi: 10.1093/emboj/16.14.4311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Llorca O., Smyth M. G., Marco S., Carrascosa J. L., Willison K. R., Valpuesta J. M. ATP binding induces large conformational changes in the apical and equatorial domains of the eukaryotic chaperonin containing TCP-1 complex. J Biol Chem. 1998 Apr 24;273(17):10091–10094. doi: 10.1074/jbc.273.17.10091. [DOI] [PubMed] [Google Scholar]
  27. Mascorro-Gallardo J. O., Covarrubias A. A., Gaxiola R. Construction of a CUP1 promoter-based vector to modulate gene expression in Saccharomyces cerevisiae. Gene. 1996 Jun 12;172(1):169–170. doi: 10.1016/0378-1119(96)00059-5. [DOI] [PubMed] [Google Scholar]
  28. Melki R., Cowan N. J. Facilitated folding of actins and tubulins occurs via a nucleotide-dependent interaction between cytoplasmic chaperonin and distinctive folding intermediates. Mol Cell Biol. 1994 May;14(5):2895–2904. doi: 10.1128/mcb.14.5.2895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Murray A. W. Cell cycle extracts. Methods Cell Biol. 1991;36:581–605. [PubMed] [Google Scholar]
  31. Nathan D. F., Vos M. H., Lindquist S. In vivo functions of the Saccharomyces cerevisiae Hsp90 chaperone. Proc Natl Acad Sci U S A. 1997 Nov 25;94(24):12949–12956. doi: 10.1073/pnas.94.24.12949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Netzer W. J., Hartl F. U. Protein folding in the cytosol: chaperonin-dependent and -independent mechanisms. Trends Biochem Sci. 1998 Feb;23(2):68–73. doi: 10.1016/s0968-0004(97)01171-7. [DOI] [PubMed] [Google Scholar]
  33. Netzer W. J., Hartl F. U. Recombination of protein domains facilitated by co-translational folding in eukaryotes. Nature. 1997 Jul 24;388(6640):343–349. doi: 10.1038/41024. [DOI] [PubMed] [Google Scholar]
  34. Pfund C., Lopez-Hoyo N., Ziegelhoffer T., Schilke B. A., Lopez-Buesa P., Walter W. A., Wiedmann M., Craig E. A. The molecular chaperone Ssb from Saccharomyces cerevisiae is a component of the ribosome-nascent chain complex. EMBO J. 1998 Jul 15;17(14):3981–3989. doi: 10.1093/emboj/17.14.3981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. Schoepfer R. The pRSET family of T7 promoter expression vectors for Escherichia coli. Gene. 1993 Feb 14;124(1):83–85. doi: 10.1016/0378-1119(93)90764-t. [DOI] [PubMed] [Google Scholar]
  37. Shevchenko A., Jensen O. N., Podtelejnikov A. V., Sagliocco F., Wilm M., Vorm O., Mortensen P., Shevchenko A., Boucherie H., Mann M. Linking genome and proteome by mass spectrometry: large-scale identification of yeast proteins from two dimensional gels. Proc Natl Acad Sci U S A. 1996 Dec 10;93(25):14440–14445. doi: 10.1073/pnas.93.25.14440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. 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]
  40. 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]
  41. 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]
  42. 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]
  43. Ursic D., Sedbrook J. C., Himmel K. L., Culbertson M. R. The essential yeast Tcp1 protein affects actin and microtubules. Mol Biol Cell. 1994 Oct;5(10):1065–1080. doi: 10.1091/mbc.5.10.1065. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Vainberg I. E., Lewis S. A., Rommelaere H., Ampe C., Vandekerckhove J., Klein H. L., Cowan N. J. Prefoldin, a chaperone that delivers unfolded proteins to cytosolic chaperonin. Cell. 1998 May 29;93(5):863–873. doi: 10.1016/s0092-8674(00)81446-4. [DOI] [PubMed] [Google Scholar]
  45. 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]
  46. 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]
  47. Weissman J. S., Kashi Y., Fenton W. A., Horwich A. L. GroEL-mediated protein folding proceeds by multiple rounds of binding and release of nonnative forms. Cell. 1994 Aug 26;78(4):693–702. doi: 10.1016/0092-8674(94)90533-9. [DOI] [PubMed] [Google Scholar]
  48. Wiedmann B., Sakai H., Davis T. A., Wiedmann M. A protein complex required for signal-sequence-specific sorting and translocation. Nature. 1994 Aug 11;370(6489):434–440. doi: 10.1038/370434a0. [DOI] [PubMed] [Google Scholar]
  49. 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]

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

RESOURCES