Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Defining the TRiC/CCT interactome links chaperonin function to stabilization of newly made proteins with complex topologies

Nature Structural & Molecular Biology volume 15, pages 1255–1262 (2008)Cite this article

Abstract

Folding within the crowded cellular milieu often requires assistance from molecular chaperones that prevent inappropriate interactions leading to aggregation and toxicity. The contribution of individual chaperones to folding the proteome remains elusive. Here we demonstrate that the eukaryotic chaperonin TRiC/CCT (TCP1-ring complex or chaperonin containing TCP1) has broad binding specificity in vitro, similar to the prokaryotic chaperonin GroEL. However, in vivo, TRiC substrate selection is not based solely on intrinsic determinants; instead, specificity is dictated by factors present during protein biogenesis. The identification of cellular substrates revealed that TRiC interacts with folding intermediates of a subset of structurally and functionally diverse polypeptides. Bioinformatics analysis revealed an enrichment in multidomain proteins and regions of β-strand propensity that are predicted to be slow folding and aggregation prone. Thus, TRiC may have evolved to protect complex protein topologies within its central cavity during biosynthesis and folding.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Principles of TRiC substrate selection in the eukaryotic cytosol.
Figure 2: Screening for the TRiC interactome in the context of translation.
Figure 3: In vivo analysis of the TRiC interactome reveals distinct kinetics of substrate flux through the chaperonin.
Figure 4: Physical and structural properties of the TRiC interactome.
Figure 5: Function of TRiC during de novo protein folding.

Similar content being viewed by others

References

  1. Frydman, J. Folding of newly translated proteins in vivo: the role of molecular chaperones. Annu. Rev. Biochem. 70, 603–647 (2001).

    Article  CAS  PubMed  Google Scholar 

  2. Young, J.C., Agashe, V.R., Siegers, K. & Hartl, F.U. Pathways of chaperone-mediated protein folding in the cytosol. Nat. Rev. Mol. Cell Biol. 5, 781–791 (2004).

    Article  CAS  PubMed  Google Scholar 

  3. Stefani, M. & Dobson, C.M. Protein aggregation and aggregate toxicity: new insights into protein folding, misfolding diseases and biological evolution. J. Mol. Med. 81, 678–699 (2003).

    Article  CAS  PubMed  Google Scholar 

  4. Ross, C.A. & Poirier, M.A. Protein aggregation and neurodegenerative disease. Nat. Med. 10, S10–S17 (2004).

    Article  PubMed  Google Scholar 

  5. Spiess, C., Meyer, A.S., Reissmann, S. & Frydman, J. Mechanism of the eukaryotic chaperonin: protein folding in the chamber of secrets. Trends Cell Biol. 14, 598–604 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Horwich, A.L., Fenton, W.A., Chapman, E. & Farr, G.W. Two families of chaperonin: physiology and mechanism. Annu. Rev. Cell Dev. Biol. 23, 115–145 (2007).

    Article  CAS  PubMed  Google Scholar 

  7. Gomez-Puertas, P., Martin-Benito, J., Carrascosa, J.L., Willison, K.R. & Valpuesta, J.M. The substrate recognition mechanisms in chaperonins. J. Mol. Recognit. 17, 85–94 (2004).

    Article  CAS  PubMed  Google Scholar 

  8. Kubota, S., Kubota, H. & Nagata, K. Cytosolic chaperonin protects folding intermediates of Gβ from aggregation by recognizing hydrophobic β-strands. Proc. Natl. Acad. Sci. USA 103, 8360–8365 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Camasses, A., Bogdanova, A., Shevchenko, A. & Zachariae, W. The CCT chaperonin promotes activation of the anaphase-promoting complex through the generation of functional Cdc20. Mol. Cell 12, 87–100 (2003).

    Article  CAS  PubMed  Google Scholar 

  10. Feldman, D.E., Thulasiraman, V., Ferreyra, R.G. & Frydman, J. Formation of the VHL-elongin BC tumor suppressor complex is mediated by the chaperonin TRiC. Mol. Cell 4, 1051–1061 (1999).

    Article  CAS  PubMed  Google Scholar 

  11. Guenther, M.G., Yu, J., Kao, G.D., Yen, T.J. & Lazar, M.A. Assembly of the SMRT-histone deacetylase 3 repression complex requires the TCP-1 ring complex. Genes Dev. 16, 3130–3135 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Won, K.A., Schumacher, R.J., Farr, G.W., Horwich, A.L. & Reed, S.I. Maturation of human cyclin E requires the function of eukaryotic chaperonin CCT. Mol. Cell. Biol. 18, 7584–7589 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Albanese, V., Yam, A.Y., Baughman, J., Parnot, C. & Frydman, J. Systems analyses reveal two chaperone networks with distinct functions in eukaryotic cells. Cell 124, 75–88 (2006).

    Article  CAS  PubMed  Google Scholar 

  14. Thulasiraman, V., Yang, C.F. & Frydman, J. In vivo newly translated polypeptides are sequestered in a protected folding environment. EMBO J. 18, 85–95 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Pappenberger, G. et al. Crystal structure of the CCTγ apical domain: implications for substrate binding to the eukaryotic cytosolic chaperonin. J. Mol. Biol. 318, 1367–1379 (2002).

    Article  CAS  PubMed  Google Scholar 

  16. Hynes, G.M. & Willison, K.R. Individual subunits of the eukaryotic cytosolic chaperonin mediate interactions with binding sites located on subdomains of β-actin. J. Biol. Chem. 275, 18985–18994 (2000).

    Article  CAS  PubMed  Google Scholar 

  17. Frydman, J., Nimmesgern, E., Ohtsuka, K. & Hartl, F.U. Folding of nascent polypeptide chains in a high molecular mass assembly with molecular chaperones. Nature 370, 111–117 (1994).

    Article  CAS  PubMed  Google Scholar 

  18. Melville, M.W., McClellan, A.J., Meyer, A.S., Darveau, A. & Frydman, J. The Hsp70 and TRiC/CCT chaperone systems cooperate in vivo to assemble the von Hippel-Lindau tumor suppressor complex. Mol. Cell. Biol. 23, 3141–3151 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Melki, R., Batelier, G., Soulie, S. & Williams, R.C. Jr. Cytoplasmic chaperonin containing TCP-1: structural and functional characterization. Biochemistry 36, 5817–5826 (1997).

    Article  CAS  PubMed  Google Scholar 

  20. Feldman, D.E., Spiess, C., Howard, D.E. & Frydman, J. Tumorigenic mutations in VHL disrupt folding in vivo by interfering with chaperonin binding. Mol. Cell 12, 1213–1224 (2003).

    Article  CAS  PubMed  Google Scholar 

  21. Rommelaere, H., De Neve, M., Melki, R., Vandekerckhove, J. & Ampe, C. The cytosolic class II chaperonin CCT recognizes delineated hydrophobic sequences in its target proteins. Biochemistry 38, 3246–3257 (1999).

    Article  CAS  PubMed  Google Scholar 

  22. King, R.W., Lustig, K.D., Stukenberg, P.T., McGarry, T.J. & Kirschner, M.W. Expression cloning in the test tube. Science 277, 973–974 (1997).

    Article  CAS  PubMed  Google Scholar 

  23. Nimmesgern, E. & Hartl, F.U. ATP-dependent protein refolding activity in reticulocyte lysate. Evidence for the participation of different chaperone components. FEBS Lett. 331, 25–30 (1993).

    Article  CAS  PubMed  Google Scholar 

  24. Jermutus, L., Ryabova, L.A. & Pluckthun, A. Recent advances in producing and selecting functional proteins by using cell-free translation. Curr. Opin. Biotechnol. 9, 534–548 (1998).

    Article  CAS  PubMed  Google Scholar 

  25. Zhao, W.M. & Fang, G. Anillin is a substrate of anaphase-promoting complex/cyclosome (APC/C) that controls spatial contractility of myosin during late cytokinesis. J. Biol. Chem. 280, 33516–33524 (2005).

    Article  CAS  PubMed  Google Scholar 

  26. Ho, Y. et al. Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry. Nature 415, 180–183 (2002).

    Article  CAS  PubMed  Google Scholar 

  27. Zhu, H. et al. Global analysis of protein activities using proteome chips. Science 293, 2101–2105 (2001).

    Article  CAS  PubMed  Google Scholar 

  28. Winzeler, E.A. et al. Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285, 901–906 (1999).

    Article  CAS  PubMed  Google Scholar 

  29. Mewes, H.W. et al. MIPS: analysis and annotation of proteins from whole genomes. Nucleic Acids Res. 32, D41–D44 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Jeong, H., Mason, S.P., Barabasi, A.L. & Oltvai, Z. N. Lethality and centrality in protein networks. Nature 411, 41–42 (2001).

    Article  CAS  PubMed  Google Scholar 

  31. Ditzel, L. et al. Crystal structure of the thermosome, the archaeal chaperonin and homolog of CCT. Cell 93, 125–138 (1998).

    Article  CAS  PubMed  Google Scholar 

  32. Srikakulam, R. & Winkelmann, D.A. Myosin II folding is mediated by a molecular chaperonin. J. Biol. Chem. 274, 27265–27273 (1999).

    Article  CAS  PubMed  Google Scholar 

  33. Chapman, E. et al. Global aggregation of newly translated proteins in an Escherichia coli strain deficient of the chaperonin GroEL. Proc. Natl. Acad. Sci. USA 103, 15800–15805 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Sambashivan, S., Liu, Y., Sawaya, M.R., Gingery, M. & Eisenberg, D. Amyloid-like fibrils of ribonuclease A with three-dimensional domain-swapped and native-like structure. Nature 437, 266–269 (2005).

    Article  CAS  PubMed  Google Scholar 

  35. Richardson, J.S. & Richardson, D.C. Natural β-sheet proteins use negative design to avoid edge-to-edge aggregation. Proc. Natl. Acad. Sci. USA 99, 2754–2759 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Gong, H., Isom, D.G., Srinivasan, R. & Rose, G.D. Local secondary structure content predicts folding rates for simple, two-state proteins. J. Mol. Biol. 327, 1149–1154 (2003).

    Article  CAS  PubMed  Google Scholar 

  37. Miller, E.J., Fischer, K.F. & Marqusee, S. Experimental evaluation of topological parameters determining protein-folding rates. Proc. Natl. Acad. Sci. USA 99, 10359–10363 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Plaxco, K.W., Simons, K.T. & Baker, D. Contact order, transition state placement and the refolding rates of single domain proteins. J. Mol. Biol. 277, 985–994 (1998).

    Article  CAS  PubMed  Google Scholar 

  39. Tam, S., Geller, R., Spiess, C. & Frydman, J. The chaperonin TRiC controls polyglutamine aggregation and toxicity through subunit-specific interactions. Nat. Cell Biol. 8, 1155–1162 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Kitamura, A. et al. Cytosolic chaperonin prevents polyglutamine toxicity with altering the aggregation state. Nat. Cell Biol. 8, 1163–1170 (2006).

    Article  CAS  PubMed  Google Scholar 

  41. Behrends, C. et al. Chaperonin TRiC promotes the assembly of polyQ expansion proteins into nontoxic oligomers. Mol. Cell 23, 887–897 (2006).

    Article  CAS  PubMed  Google Scholar 

  42. Reissmann, S., Parnot, C., Booth, C.R., Chiu, W. & Frydman, J. Essential function of the built-in lid in the allosteric regulation of eukaryotic and archaeal chaperonins. Nat. Struct. Mol. Biol. 14, 432–440 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Richter, K. & Buchner, J. Hsp90: chaperoning signal transduction. J. Cell. Physiol. 188, 281–290 (2001).

    Article  CAS  PubMed  Google Scholar 

  44. Dekker, C. et al. The interaction network of the chaperonin CCT. EMBO J. 27, 1827–1839 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Vinh, D.B. & Drubin, D.G. A yeast TCP-1-like protein is required for actin function in vivo. Proc. Natl. Acad. Sci. USA 91, 9116–9120 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Yaffe, M.B. et al. TCP1 complex is a molecular chaperone in tubulin biogenesis. Nature 358, 245–248 (1992).

    Article  CAS  PubMed  Google Scholar 

  47. Kerner, M.J. et al. Proteome-wide analysis of chaperonin-dependent protein folding in Escherichia coli. Cell 122, 209–220 (2005).

    Article  CAS  PubMed  Google Scholar 

  48. Hellman, U., Wernstedt, C., Gonez, J. & Heldin, C.H. Improvement of an “in-gel” digestion procedure for the micropreparation of internal protein fragments for amino acid sequencing. Anal. Biochem. 224, 451–455 (1995).

    Article  CAS  PubMed  Google Scholar 

  49. Labbe, S. & Thiele, D.J. Copper ion inducible and repressible promoter systems in yeast. Methods Enzymol. 306, 145–153 (1999).

    Article  CAS  PubMed  Google Scholar 

  50. Kopp, J. & Schwede, T. The SWISS-MODEL Repository of annotated three-dimensional protein structure homology models. Nucleic Acids Res. 32, D230–D234 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. 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 89, 927–937 (1997).

    Article  CAS  PubMed  Google Scholar 

  52. Frydman, J. et al. Function in protein folding of TRiC, a cytosolic ring complex containing TCP-1 and structurally related subunits. EMBO J. 11, 4767–4778 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. McCallum, C.D., Do, H., Johnson, A.E. & Frydman, J. The interaction of the chaperonin tailless complex polypeptide 1 (TCP1) ring complex (TRiC) with ribosome-bound nascent chains examined using photo-cross-linking. J. Cell Biol. 149, 591–602 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Hong, S. et al. Type D retrovirus Gag polyprotein interacts with the cytosolic chaperonin TRiC. J. Virol. 75, 2526–2534 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Pijnappel, W.W. et al. The S. cerevisiae SET3 complex includes two histone deacetylases, Hos2 and Hst1, and is a meiotic-specific repressor of the sporulation gene program. Genes Dev. 15, 2991–3004 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Krogh, A., Larsson, B., von Heijne, G. & Sonnhammer, E.L. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J. Mol. Biol. 305, 567–580 (2001).

    Article  CAS  PubMed  Google Scholar 

  57. Wolf, Y.I., Brenner, S.E., Bash, P.A. & Koonin, E.V. Distribution of protein folds in the three superkingdoms of life. Genome Res. 9, 17–26 (1999).

    CAS  PubMed  Google Scholar 

  58. Kyte, J. & Doolittle, R.F. A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157, 105–132 (1982).

    Article  CAS  PubMed  Google Scholar 

  59. Jones, D.T. Protein secondary structure prediction based on position-specific scoring matrices. J. Mol. Biol. 292, 195–202 (1999).

    Article  CAS  PubMed  Google Scholar 

  60. Press, W.H., Flannery, B.P., Teukolsky, S.A. & Vetterling, W.T. Numerical Recipes in C: The Art of Scientific Computing (Cambridge University Press, New York, NY, 1992).

    Google Scholar 

Download references

Acknowledgements

The authors thank members of the Frydman laboratory and R. Andino for comments and discussion. The cDNA library was a generous gift from W. Zhao and G. Fang (Department of Biology, Stanford University). We thank W. Harper (Department of Pathology, Harvard Medical School), T. Kinzy (Department of Molecular Genetics & Microbiology, Rutgers University), and Michael Rexach (Department of Biological Sciences, Stanford University) for gifts of plasmids and antibodies. This work was supported by grants from the US National Institutes of Health and the W.M. Keck Foundation.

Author information

Author notes

  1. Alice Y Yam & Yu Xia

    Present address: Present addresses: Novartis Vaccines and Diagnostics, Inc., 4560 Horton Street, M/S 4.3, Emeryville, California 94608, USA (A.Y.Y.); Department of Chemistry, 44 Cummington Street, Boston University, Boston, Massachusetts 02215, USA (Y.X.).,

Authors and Affiliations

  1. Department of Biology and BioX Program, E200A James Clark Center, 318 Campus Drive, Stanford University, Stanford, 94043, California, USA

    Alice Y Yam & Judith Frydman

  2. Department of Molecular Biophysics & Biochemistry, PO Box 208114, Yale University, New Haven, 06520, Connecticut, USA

    Yu Xia & Mark Gerstein

  3. Department of Pharmaceutical Chemistry, 600 16th Street, MC 2240, Genentech Hall, Suite N472A, University of California at San Francisco, San Francisco, 94143, California, USA

    Hen-Tzu Jill Lin & Alma Burlingame

Authors
  1. Alice Y Yam
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yu Xia
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hen-Tzu Jill Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Alma Burlingame
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Mark Gerstein
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Judith Frydman
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.F. directed the project; A.Y.Y. and J.F. designed the project, analyzed the data and wrote the manuscript; A.Y.Y. carried out all experiments; H.-T.J.L. and A.B. carried out MS analysis; and Y.X. and M.G. carried out bioinformatics data analysis and contributed to the manuscript.

Corresponding author

Correspondence to Judith Frydman.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4 and Supplementary Table 1 (PDF 1814 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Yam, A., Xia, Y., Lin, HT. et al. Defining the TRiC/CCT interactome links chaperonin function to stabilization of newly made proteins with complex topologies. Nat Struct Mol Biol 15, 1255–1262 (2008). https://doi.org/10.1038/nsmb.1515

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.1515

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing