Review: cellular substrates of the eukaryotic chaperonin TRiC/CCT

The TCP-1 ring complex (TRiC; also called CCT, for chaperonin containing TCP-1) is a large (approximately 900 kDa) multisubunit complex that mediates protein folding in the eukaryotic cytosol. The physiological substrate spectrum of TRiC is still poorly defined. Genetic and biochemical data show that it is required for the folding of the cytoskeletal proteins actin and tubulin. Recent years have witnessed a steady stream of reports that describe other proteins that require TRiC for proper folding. Furthermore, analysis of the transit of newly synthesized proteins through TRiC in intact cells suggests that the chaperonin contributes to the folding of a distinct subset of cellular proteins. Here we review the current understanding of a role for TRiC in the folding of newly synthesized polypeptides, with a focus on some of the individual proteins that require TRiC.     

Mechanism of the eukaryotic chaperonin: protein folding in the chamber of secrets

Chaperonins are key components of the cellular chaperone machinery. These large, cylindrical complexes contain a central cavity that binds to unfolded polypeptides and sequesters them from the cellular environment. Substrate folding then occurs in this central cavity in an ATP-dependent manner. The eukaryotic chaperonin TCP-1 ring complex (TRiC, also called CCT) is indispensable for cell survival because the folding of an essential subset of cytosolic proteins requires TRiC, and this function cannot be substituted by other chaperones. This specificity indicates that TRiC has evolved structural and mechanistic features that distinguish it from other chaperones. Although knowledge of this unique complex is in its infancy, we review recent advances that open the way to understanding the secrets of its folding chamber.     

Type D retrovirus Gag polyprotein interacts with the cytosolic chaperonin TRiC

The carboxy terminus-encoding portion of the gag gene of Mason-Pfizer monkey virus (M-PMV), the prototype immunosuppressive primate type D retrovirus, encodes a 36-amino-acid, proline-rich protein domain that, in the mature virion, becomes the p4 capsid protein. The p4 domain has no known role in M-PMV replication. We found that two mutants with premature termination codons that remove half or all of the p4 domain produced lower levels of stable Gag protein and of self-assembled capsids. Interestingly, yeast two-hybrid screening revealed that p4 specifically interacted with TCP-1gamma, a subunit of the chaperonin TRiC (TCP-1 ring complex). TRiC is a cytosolic chaperonin that is known to be involved in both folding and subunit assembly of a variety of cellular proteins. TCP-1gamma also associated with high specificity with the M-PMV pp24/16-p12 domain and human immunodeficiency virus p6. Moreover, in cells, Gag polyprotein associated with the TRiC chaperonin complex and this association depended on ATP hydrolysis. In the p4 truncation mutants, the Gag-TRiC association was significantly reduced. These results strongly suggest that cytosolic chaperonin TRiC is involved in Gag folding and/or capsid assembly. We propose that TRiC associates transiently with nascent M-PMV Gag molecules to assist in their folding. Consequently, properly folded Gag molecules carry out the intermolecular interactions involved in self-assembly of the immature capsid.     

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

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.     

Conformational rearrangements of tail-less complex polypeptide 1 (TCP-1) ring complex (TRiC)-bound actin

The mechanism of chaperonins is still under intense investigation. Earlier studies by others and us on the bacterial chaperonin GroEL points to an active role of chaperonins in unfolding the target protein during initial binding. Here, a natural eukaryotic chaperonin system [tail-less complex polypeptide 1 (TCP-1) ring complex (TRiC) and its target protein actin] was investigated to determine if the active participation of the chaperonin in the folding process is evolutionary-conserved. Using fluorescence resonance energy transfer (FRET) measurements on four distinct doubly fluorescein-labeled variants of actin, we have obtained a fairly detailed map of the structural rearrangements that occur during the TRiC-actin interaction. The results clearly show that TRiC has an active role in rearranging the bound actin molecule. The target is stretched as a consequence of binding to TRiC and further rearranged in a second step as a consequence of ATP binding; i.e., the mechanism of chaperonins is conserved during evolution.     

Closing the folding chamber of the eukaryotic chaperonin requires the transition state of ATP hydrolysis

Chaperonins use ATPase cycling to promote conformational changes leading to protein folding. The prokaryotic chaperonin GroEL requires a cofactor, GroES, which serves as a "lid" enclosing substrates in the central cavity and confers an asymmetry on GroEL required for cooperative transitions driving the reaction. The eukaryotic chaperonin TRiC/CCT does not have such a cofactor but appears to have a "built-in" lid. Whether this seemingly symmetric chaperonin also operates through an asymmetric cycle is unclear. We show that unlike GroEL, TRiC does not close its lid upon nucleotide binding, but instead responds to the trigonal-bipyramidal transition state of ATP hydrolysis. Further, nucleotide analogs inducing this transition state confer an asymmetric conformation on TRiC. Similar to GroEL, lid closure in TRiC confines the substrates in the cavity and is essential for folding. Understanding the distinct mechanisms governing eukaryotic and bacterial chaperonin function may reveal how TRiC has evolved to fold specific eukaryotic proteins.     

The cotranslational contacts between ribosome-bound nascent polypeptides and the subunits of the hetero-oligomeric chaperonin TRiC probed by photocross-linking

The hetero-oligomeric eukaryotic chaperonin TRiC (TCP-1-ring complex, also called CCT) interacts cotranslationally with a diverse subset of newly synthesized proteins, including actin, tubulin, and luciferase, and facilitates their correct folding. A photocross-linking approach has been used to map the contacts between individual chaperonin subunits and ribosome-bound nascent chains of increasing length. Whereas a cryo-EM study suggests that chemically denatured actin interacts with only two TRiC subunits (delta and either beta or epsilon), actin and luciferase chains photocross-link to at least six TRiC subunits (alpha, beta, delta, epsilon, xi, and theta) at different stages of translation. Furthermore, the photocross-linking of actin, but not luciferase, nascent chains to TRiC subunits zeta and theta was length-dependent. In addition, a single photoreactive probe incorporated at a unique site in actin nascent chains of different lengths reacted covalently with multiple TRiC subunits, thereby indicating that the nascent chain samples the polypeptide binding sites of different subunits. We conclude that elongating actin and luciferase nascent chains contact multiple TRiC subunits upon emerging from the ribosome, and that the TRiC subunits contacted by nascent actin change as it elongates and starts to fold.     

Actin mutations in hypertrophic and dilated cardiomyopathy cause inefficient protein folding and perturbed filament formation

Hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) are the most common hereditary cardiac conditions. Both are frequent causes of sudden death and are often associated with an adverse disease course. Alpha-cardiac actin is one of the disease genes where different missense mutations have been found to cause either HCM or DCM. We have tested the hypothesis that the protein-folding pathway plays a role in disease development for two actin variants associated with DCM and six associated with HCM. Based on a cell-free coupled translation assay the actin variants could be graded by their tendency to associate with the chaperonin TCP-1 ring complex/chaperonin containing TCP-1 (TRiC/CCT) as well as their propensity to acquire their native conformation. Some variant proteins are completely stalled in a complex with TRiC and fail to fold into mature globular actin and some appear to fold as efficiently as the wild-type protein. A fraction of the translated polypeptide became ubiquitinated and detergent insoluble. Variant actin proteins overexpressed in mammalian cell lines fail to incorporate into actin filaments in a manner correlating with the degree of misfolding observed in the cell-free assay; ranging from incorporation comparable to wild-type actin to little or no incorporation. We propose that effects of mutations on folding and fiber assembly may play a role in the molecular disease mechanism.     

Trigger factor is involved in GroEL-dependent protein degradation in Escherichia coli and promotes binding of GroEL to unfolded proteins.

In Escherichia coli, the molecular chaperones of hsp60/hsp10 (GroEL/GroES) families are required not only for protein folding but also for the rapid degradation of certain abnormal proteins. The rate-limiting step in the degradation of the fusion protein CRAG by protease ClpP appears to be the formation of a complex with GroEL. We have isolated these complexes and found that each GroEL 14mer contained a short-lived fragment of CRAG plus a 50 kDa polypeptide, which we identified by sequencing and immunological methods as Trigger Factor (TF). Upon ATP addition, GroEL and TF dissociated together from CRAG but remained tightly associated with each other even upon gel filtration. TF was originally proposed to function in protein translocation across membranes but altering cellular content of TF did not affect this process in vivo. By contrast, low levels of TF expression markedly reduced CRAG degradation, while an overproduction of TF greatly stimulated this process. Furthermore, in extracts of cells expressing high levels of TF, the capacity of GroEL to bind to CRAG is greatly increased. Overproduction of TF also stimulated GroEL's ability to bind to other unfolded proteins (fetuin and histone). Thus, TF is a rate-limiting factor for CRAG degradation; it appears to regulate GroEL function and to promote the formation of TF-GroEL-CRAG complexes which are critical for proteolysis.     

Dual function of protein confinement in chaperonin-assisted protein folding.

The GroEL/GroES chaperonin system mediates the folding of a range of newly synthesized polypeptides in the bacterial cytosol. Using a rapid biotin-streptavidin-based inhibition of chaperonin function, we show that the cage formed by GroEL and its cofactor GroES can have a dual role in promoting folding. First, enclosure of nonnative protein in the GroEL:GroES complex is essential for folding to proceed unimpaired by aggregation. Second, folding inside the cage can be significantly faster than folding in free solution, independently of ATP-driven cycles of GroES binding and release. This suggests that confinement of unfolded protein in the narrow hydrophilic space of the chaperonin cage smoothes the energy landscape for the folding of some proteins, increasing the flux of folding intermediates toward the native state.     

In vivo newly translated polypeptides are sequestered in a protected folding environment.

Molecular chaperones play a fundamental role in cellular protein folding. Using intact mammalian cells we examined the contribution of cytosolic chaperones to de novo folding. A large fraction of newly translated polypeptides associate transiently with Hsc70 and the chaperonin TRiC/CCT during their biogenesis. The substrate repertoire observed for Hsc70 and TRiC is not identical: Hsc70 interacts with a wide spectrum of polypeptides larger than 20 kDa, while TRiC associates with a diverse set of proteins between 30 and 60 kDa. Overexpression of a bacterial chaperonin 'trap' that irreversibly captures unfolded polypeptides did not interrupt the productive folding pathway. The trap was unable to bind newly translated polypeptides, indicating that folding in mammalian cells occurs without the release of non-native folding intermediates into the bulk cytosol. We conclude that de novo protein folding occurs in a protected environment created by a highly processive chaperone machinery and is directly coupled to translation.     

Design of a molecular chaperone-assisted protein folding bioreactor

Escherichia coli molecular chaperone GroEL and co-chaperone GroES are well known to assist the folding/refolding of a diverse range of substrate proteins. Despite this, there have been relatively few reports of the GroEL/GroES molecular chaperone system being used as a biotechnology tool for protein folding/refolding. In this paper, a solution-phase protein folding bioreactor is described that involves the complete GroEL/GroES system. The main features of this bioreactor are the use of a stirred-cell concentrator fitted with a 100 kDa molecular weight cutoff membrane and an attached buffer reservoir. This bioreactor system was used successfully for assisted-batch refolding of guanidinium chloride (Gu-HCl) unfolded mitochondrial malate dehydrogenase (mMDH). We believe that protein folding bioreactor systems of this type could have wide potential utility for the folding/refolding of unfolded protein substrates.     

Human CCT4 and CCT5 Chaperonin Subunits Expressed in Escherichia coli Form Biologically Active Homo-oligomers

Chaperonins are a family of chaperones that encapsulate their substrates and assist their folding in an ATP-dependent manner. The ubiquitous eukaryotic chaperonin, TCP-1 ring complex (TRiC), is a hetero-oligomeric complex composed of two rings, each formed from eight different CCT (chaperonin containing TCP-1) subunits. Each CCT subunit may have distinct substrate recognition and ATP hydrolysis properties. We have expressed each human CCT subunit individually in Escherichia coli to investigate whether they form chaperonin-like double ring complexes. CCT4 and CCT5, but not the other six CCT subunits, formed high molecular weight complexes within the E. coli cells that sedimented about 20S in sucrose gradients. When CCT4 and CCT5 were purified, they were both organized as two back-to-back rings of eight subunits each, as seen by negative stain and cryo-electron microscopy. This morphology is consistent with that of the hetero-oligomeric double-ring TRiC purified from bovine testes and HeLa cells. Both CCT4 and CCT5 homo-oligomers hydrolyzed ATP at a rate similar to human TRiC and were active as assayed by luciferase refolding and human γD-crystallin aggregation suppression and refolding. Thus, both CCT4 and CCT5 homo-oligomers have the property of forming 8-fold double rings absent the other subunits, and these complexes carry out chaperonin reactions without other partner subunits.     

The substrate recognition mechanisms in chaperonins

Chaperonins are a family of proteins devoted to assisting the folding of other proteins. They are large oligomers assembled into ring structures that enclose a cavity in which folding takes place. For this process to occur, the chaperonin must first recognize and interact with the unfolded polypeptide, then undergo a conformational change upon nucleotide binding that results in the closure of the cavity which in turn mediates the folding reaction inside the cavity. Although this general mechanism seems to apply to every chaperonin studied so far, there exist two different modes of interaction between the chaperonin and the substrate. The first occurs mainly through the interaction between the exposed hydrophobic residues of the unfolded polypeptides and those of the chaperonin substrate binding site, as elucidated for the chaperonin GroEL from E. coli. The second type of mechanism has been described so far only for the cytosolic chaperonin CCT (Chaperonin Containing TCP-1) and here the interaction seems to be of a more specific nature, involving charged and polar residues in both the chaperonin and the substrate, which interacts with CCT in a structured, quasi-native conformation.     

Crystal structure of the CCTgamma apical domain: implications for substrate binding to the eukaryotic cytosolic chaperonin

The chaperonin containing TCP-1 (CCT, also known as TRiC) is the only member of the chaperonin family found in the cytosol of eukaryotes. Like other chaperonins, it assists the folding of newly synthesised proteins. It is, however, unique in its specificity towards only a small subset of non-native proteins. We determined two crystal structures of mouse CCTgamma apical domain at 2.2 A and 2.8 A resolution. They reveal a surface patch facing the inside of the torus that is highly evolutionarily conserved and specific for the CCTgamma apical domain. This putative substrate-binding region consists of predominantly positively charged side-chains. It suggests that the specificity of this apical domain towards its substrate, partially folded tubulin, is conferred by polar and electrostatic interactions. The site and nature of substrate interaction are thus profoundly different between CCT and its eubacterial homologue GroEL, consistent with their different functions in general versus specific protein folding assistance.     

The role of molecular chaperones in human misfolding diseases

Human misfolding diseases arise when proteins adopt non-native conformations that endow them with a tendency to aggregate and form intra- and/or extra-cellular deposits. Molecular chaperones, such as Hsp70 and TCP-1 Ring Complex (TRiC)/chaperonin containing TCP-1 (CCT), have been implicated as potent modulators of misfolding disease. These chaperones suppress toxicity of disease proteins and modify early events in the aggregation process in a cooperative and sequential manner reminiscent of their functions in de novo protein folding. Further understanding of the role of Hsp70, TRiC, and other chaperones in misfolding disease is likely to provide important insight into basic pathomechanistic principles that could potentially be exploited for therapeutic purposes.     

Characterisation of a GroEL Single-Ring Mutant that Supports Growth of Escherichia coli and Has GroES-Dependent ATPase Activity

Binding and folding of substrate proteins by the molecular chaperone GroEL alternates between its two seven-membered rings in an ATP-regulated manner. The association of ATP and GroES to a polypeptide-bound ring of GroEL encapsulates the folding proteins in the central cavity of that ring (cis ring) and allows it to fold in a protected environment where the risk of aggregation is reduced. ATP hydrolysis in the cis ring changes the potentials within the system such that ATP binding to the opposite (trans) ring triggers the release of all ligands from the cis ring of GroEL through a complex network of allosteric communication between the rings. Inter-ring allosteric communication thus appears indispensable for the function of GroEL, and an engineered single-ring version (SR1) cannot substitute for GroEL in vivo. We describe here the isolation and characterisation of an active single-ring form of the GroEL protein (SR-A92T), which has an exceptionally low ATPase activity that is strongly stimulated by the addition of GroES. Dissection of the kinetic pathway of the ATP-induced structural changes in this active single ring can be explained by the fact that the mutation effectively blocks progression through the full allosteric pathway of the GroEL reaction cycle, thus trapping an early allosteric intermediate. Addition of GroES is able to overcome this block by binding this intermediate and pulling the allosteric pathway to completion via mass action, explaining how bacterial cells expressing this protein as their only chaperonin are viable.     

Triggering protein folding within the GroEL-GroES complex

The folding of many proteins depends on the assistance of chaperonins like GroEL and GroES and involves the enclosure of substrate proteins inside an internal cavity that is formed when GroES binds to GroEL in the presence of ATP. Precisely how assembly of the GroEL-GroES complex leads to substrate protein encapsulation and folding remains poorly understood. Here we use a chemically modified mutant of GroEL (EL43Py) to uncouple substrate protein encapsulation from release and folding. Although EL43Py correctly initiates a substrate protein encapsulation reaction, this mutant stalls in an intermediate allosteric state of the GroEL ring, which is essential for both GroES binding and the forced unfolding of the substrate protein. This intermediate conformation of the GroEL ring possesses simultaneously high affinity for both GroES and non-native substrate protein, thus preventing escape of the substrate protein while GroES binding and substrate protein compaction takes place. Strikingly, assembly of the folding-active GroEL-GroES complex appears to involve a strategic delay in ATP hydrolysis that is coupled to disassembly of the old, ADP-bound GroEL-GroES complex on the opposite ring.