Human TRiC complex purified from HeLa cells contains all eight CCT subunits and is active in vitro
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Archaeal and eukaryotic cytosols contain group II chaperonins, which have a double-barrel structure and fold proteins inside a cavity in an ATP-dependent manner. The most complex of the chaperonins, the eukaryotic TCP-1 ring complex (TRiC), has eight different subunits, chaperone containing TCP-1 (CCT1–8), that are arranged so that there is one of each subunit per ring. Aspects of the structure and function of the bovine and yeast TRiC have been characterized, but studies of human TRiC have been limited. We have isolated and purified endogenous human TRiC from HeLa suspension cells. This purified human TRiC contained all eight CCT subunits organized into double-barrel rings, consistent with what has been found for bovine and yeast TRiC. The purified human TRiC is active as demonstrated by the luciferase refolding assay. As a more stringent test, the ability of human TRiC to suppress the aggregation of human γD-crystallin was examined. In addition to suppressing off-pathway aggregation, TRiC was able to assist the refolding of the crystallin molecules, an activity not found with the lens chaperone, α-crystallin. Additionally, we show that human TRiC from HeLa cell lysate is associated with the heat shock protein 70 and heat shock protein 90 chaperones. Purification of human endogenous TRiC from HeLa cells will enable further characterization of this key chaperonin, required for the reproduction of all human cells.
KeywordsTRiC CCT Chaperonin Crystallin Protein folding
The authors thank Dr. Kate Moreau and Daniel Goulet for their helpful discussions. This work was funded by NIH Roadmap grant EY016525 and NEI grant EY015834.
- Cong Y, Baker ML, Jakana J, Woolford D, Miller EJ, Reissmann S, Kumar RN, Redding-Johanson AM, Batth TS, Mukhopadhyay A, Ludtke SJ, Frydman J, Chiu W (2010) 4.0-A resolution cryo-EM structure of the mammalian chaperonin TRiC/CCT reveals its unique subunit arrangement. Proc Natl Acad Sci USA 107(11):4967–4972PubMedCrossRefGoogle Scholar
- Dekker C, Roe SM, McCormack EA, Beuron F, Pearl LH, Willison KR (2011) The crystal structure of yeast CCT reveals intrinsic asymmetry of eukaryotic cytosolic chaperonins. EMBO J 30(15):3078–3090Google Scholar
- Kampinga HH, Hageman J, Vos MJ, Kubota H, Tanguay RM, Bruford EA, Cheetham ME, Chen B, Hightower LE (2009) Guidelines for the nomenclature of the human heat shock proteins. Cell Stress Chaperones 14:105–111Google Scholar
- Muñoz IG, Yébenes H, Zhou M, Mesa P, Serna M, Park AY, Bragado-Nilsson E, Beloso A, de Cárcer G, Malumbres M, Robinson CV, Valpuesta JM, Montoya G (2011) Crystal structure of the open conformation of the mammalian chaperonin CCT in complex with tubulin. Nat Struct Mol Biol 18(1):14–19PubMedCrossRefGoogle Scholar
- Zhang Q, Yu D, Seo S, Stone EM, Sheffield VC (2012) Intrinsic protein-protein interaction mediated and chaperonin assisted sequential assembly of a stable Bardet Biedl syndrome protein complex, the BBSome. J Biol Chem 287(24):20625–20635Google Scholar