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Human TRiC complex purified from HeLa cells contains all eight CCT subunits and is active in vitro

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Cell Stress and Chaperones Aims and scope

Abstract

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.

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Acknowledgments

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.

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  1. Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Ave., 68-330, Cambridge, MA, 02139, USA

    Kelly M. Knee, Oksana A. Sergeeva & Jonathan A. King

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  1. Kelly M. Knee
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  2. Oksana A. Sergeeva
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  3. Jonathan A. King
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Correspondence to Jonathan A. King.

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Knee, K.M., Sergeeva, O.A. & King, J.A. Human TRiC complex purified from HeLa cells contains all eight CCT subunits and is active in vitro. Cell Stress and Chaperones 18, 137–144 (2013). https://doi.org/10.1007/s12192-012-0357-z

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  • DOI: https://doi.org/10.1007/s12192-012-0357-z

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