The Molecular Architecture of the Eukaryotic Chaperonin TRiC/CCT

Abstract

Summary TRiC/CCT is a highly conserved and essential chaperonin that uses ATP cycling to facilitate folding of approximately 10% of the eukaryotic proteome. This 1 MDa hetero-oligomeric complex consists of two stacked rings of eight paralogous subunits each. Previously proposed TRiC models differ substantially in their subunit arrangements and ring register. Here, we integrate chemical crosslinking, mass spectrometry and combinatorial modeling to reveal the definitive subunit arrangement of TRiC. In vivo disulfide mapping provided additional validation for the crosslinking-derived arrangement as the definitive TRiC topology. This subunit arrangement allowed the refinement of a structural model using existing X-ray diffraction data. The new structure explains all available crosslink experiments, provides a rationale for previously unexplained structural features and reveals a surprising asymmetry of charges within the chaperonin folding chamber.

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

“…While TRiC has been readily purified endogenously from bovine testes (Frydman et al 1992;Leitner et al 2012) and pseudo-exogenously from yeast (Leitner et al 2012;Pappenberger et al 2006), purification of human TRiC has been limited. Expression of all eight subunits exogenously from the cloned genes is difficult.…”

“…Purification of endogenous TRiC from rabbit reticulocytes has also been effective (Frydman et al 1994;Gao et al 1992;Nimmesgern and Hartl 1993;Norcum 1996). More recently, purification of exogenously tagged yeast TRiC in yeast has been developed (Dekker et al 2011;Pappenberger et al 2006), along with purification of endogenous yeast TRiC by exogenously tagging an interacting protein (Leitner et al 2012). Co-expression of all eight human CCT subunits in baby hamster kidney cells has been attempted but resulted in very low yields (Machida et al 2012).…”

“…However, a recent novel mass spectrometry method established that bovine TRiC purified from testes tissue and yeast TRiC purified via an interacting protein had the same arrangement with CCT2 and CCT6 forming the homotypic contacts (Leitner et al 2012). This does not rule out that other CCT subunit arrangements of TRiC may exist, especially in different tissues and at different developmental stages.…”

Folding of large multidomain proteins by partial encapsulation in the chaperonin TRiC/CCT

“…Step 3, the C-terminal domain is encapsulated by TRiC upon ATP hydrolysis and is induced to fold in the specialized physical environment of the chaperonin cavity (23). The irislike closing mechanism allows the flexible interdomain linker to protrude through the apical pore.…”

“…The TRiC chaperonin complex consists of eight distinct, paralogous subunits per ring, which are arranged in a precise orientation (22,23). These subunits have been highly conserved during evolution from a simpler archaeal precursor (24).…”