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

Abstract

The eukaryotic chaperonin, TRiC/CCT (TRiC, TCP-1 ring complex; CCT, chaperonin containing TCP-1), uses a built-in lid to mediate protein folding in an enclosed central cavity. Recent structural data suggest an effective size limit for the TRiC folding chamber of ∼70 kDa, but numerous chaperonin substrates are substantially larger. Using artificial fusion constructs with actin, an obligate chaperonin substrate, we show that TRiC can mediate folding of large proteins by segmental or domain-wise encapsulation. Single or multiple protein domains up to ∼70 kDa are stably enclosed by stabilizing the ATP-hydrolysis transition state of TRiC. Additional domains, connected by flexible linkers that pass through the central opening of the folding chamber, are excluded and remain accessible to externally added protease. Experiments with the physiological TRiC substrate hSnu114, a 109-kDa multidomain protein, suggest that TRiC has the ability to recognize domain boundaries in partially folded intermediates. In the case of hSnu114, this allows the selective encapsulation of the C-terminal ∼45-kDa domain and segments thereof, presumably reflecting a stepwise folding mechanism. The capacity of the eukaryotic chaperonin to overcome the size limitation of the folding chamber may have facilitated the explosive expansion of the multidomain proteome in eukaryotes.folding cage | molecular chaperone | Snu114 homolog | 116 kDa U5 small nuclear ribonucleoprotein component

Chaperonin TRiC/CCT Modulates the Folding and Activity of Leukemogenic Fusion Oncoprotein AML1-ETO

“…4A), and the depleted lysate remarkably lost its ability to produce AML1-ETO and STAT3 (23). On the other hand, ␤-actin did not show a significant change in protein expression (36) (Fig. 4B).…”

Essential Role of the Chaperonin CCT in Rod Outer Segment Biogenesis

“…The structural and physical features of these soluble proteins, with the predicted bpropeller (BBS2 and BBS7) and plekstrin homology (BBS5) domains, 20 are common among the bona fide CCT substrates, all of which are soluble proteins, often containing complex domain topology and WD-repeat domains that fold into a bpropeller structure. [3][4][5][6]36 It is plausible that BBS2, BBS5, and BBS7 are substrate proteins of CCT, since CCT suppression results in posttranslational destabilization of these proteins. Because BBS2 and BBS7 are the components of the core complex (BBS2-BBS7-BBS9) initiating the assembly of the BBSome, 23 their simultaneous decrease in rods would be expected to have a strong impact on the BBSome.…”

The Mechanism and Function of Group II Chaperonins

“…The potential exists for proteins to be enclosed co-translationally, or for specific domains to be sequestered within the chamber while the remainder of the protein sticks out into the cytosol. The latter has been demonstrated to occur with TRiC in in vitro translation systems using several fusions between actin and fluorescent proteins as well a natural substrate, hSnu114, which exceeds the chamber size [111]. There, encapsulation was monitored by the pattern of proteolytic fragments produced under different nucleotide conditions.…”

“…Combining this new appreciation for differential substrate recognition [108] , the demonstration of substrate folding on a per-domain basis [111], and the knowledge that there is an asymmetric utilization of nucleotide among the CCT subunits [59] presents an exciting picture of how a substrate may be released into the central chamber and folded upon ATP hydrolysis (Figure 5). With the apical domains possessing specificity for a particular sequence, each substrate would be bound in an orientation defined by the presence and arrangement of different recognition motifs within its sequence.…”