The Molecular Chaperone CCT Sequesters Gelsolin and Protects it from Cleavage by Caspase-3

The actin filament severing and capping protein gelsolin plays an important role in modulation of actin filament dynamics by influencing the number of actin filament ends. During apoptosis, gelsolin becomes constitutively active due to cleavage by caspase-3. In non-apoptotic cells gelsolin is activated by the binding of Ca²⁺. This activated form of gelsolin binds to, but is not a folding substrate of the molecular chaperone CCT/TRiC. Here we demonstrate that in vitro, gelsolin is protected from cleavage by caspase-3 in the presence of CCT. Cryoelectron microscopy and single particle 3D reconstruction of the CCT:gelsolin complex reveals that gelsolin is located in the interior of the chaperonin cavity, with a placement distinct from that of the obligate CCT folding substrates actin and tubulin. In cultured mouse melanoma B16F1 cells, gelsolin co-localises with CCT upon stimulation of actin dynamics at peripheral regions during lamellipodia formation. These data indicate that localised sequestration of gelsolin by CCT may provide spatial control of actin filament dynamics.     

Eukaryotic chaperonin containing T-complex polypeptide 1 interacts with filamentous actin and reduces the initial rate of actin polymerization in vitro

We have previously observed that subunits of the chaperonin required for actin production (type-II chaperonin containing T-complex polypeptide 1 [CCT]) localize at sites of microfilament assembly. In this article we extend this observation by showing that substantially substoichiometric CCT reduces the initial rate of pyrene-labeled actin polymerization in vitro where eubacterial chaperonin GroEL had no such effect. CCT subunits bound selectively to F-actin in cosedimentation assays, and CCT reduced elongation rates from both purified actin filament "seeds" and the short and stabilized, minus-end blocked filaments in erythrocyte membrane cytoskeletons. These observations suggest CCT might remain involved in biogenesis of the actin cytoskeleton, by acting at filament (+) ends, beyond its already well-established role in producing new actin monomers.     

Subunits of the chaperonin CCT interact with F-actin and influence cell shape and cytoskeletal assembly

The integrity of the cytoskeleton is closely linked to the oligomeric chaperonin containing TCP-1 (CCT) via the folding requirements of actin and tubulin, but the role of CCT in cytoskeletal organization remains unclear. We address this issue by analyzing the effects of targeting CCT subunits via siRNA and assessing their location/assembly state in cultured mammalian cells. Reducing levels of individual CCT subunits implicates CCT? in influencing cell shape and reduced levels of this subunit limit the cells' ability to recover from microfilament depolymerization. Conversely, cells displayed enhanced microtubule regrowth when CCT subunit levels were altered by siRNA. Some CCT subunits co-localize with F-actin, whilst all are predominantly monomeric in extracts enriched for the cytoskeleton. This provides compelling evidence that some CCT subunits as monomers can influence cytoskeletal organization/polymerization. Therefore the activity of CCT may well extend beyond the folding of newly synthesized polypeptides, representing a novel function for CCT subunits distinct from their role in the CCT oligomer.     

PhLP3 modulates CCT-mediated actin and tubulin folding via ternary complexes with substrates

Many ATP-dependent molecular chaperones, including Hsp70, Hsp90, and the chaperonins GroEL/Hsp60, require cofactor proteins to regulate their ATPase activities and thus folding functions in vivo. One conspicuous exception has been the eukaryotic chaperonin CCT, for which no regulator of its ATPase activity, other than non-native substrate proteins, is known. We identify the evolutionarily conserved PhLP3 (phosducin-like protein 3) as a modulator of CCT function in vitro and in vivo. PhLP3 binds CCT, spanning the cylindrical chaperonin cavity and contacting at least two subunits. When present in a ternary complex with CCT and an actin or tubulin substrate, PhLP3 significantly diminishes the chaperonin ATPase activity, and accordingly, excess PhLP3 perturbs actin or tubulin folding in vitro. Most interestingly, however, the Saccharomyces cerevisiae PhLP3 homologue is required for proper actin and tubulin function. This cellular role of PhLP3 is most apparent in a strain that also lacks prefoldin, a chaperone that facilitates CCT-mediated actin and tubulin folding. We propose that the antagonistic actions of PhLP3 and prefoldin serve to modulate CCT activity and play a key role in establishing a functional cytoskeleton in vivo.     

真核细胞伴侣素CCT及其与细胞骨架的关系

CCT(the chaperonin containing tailless complex polypeptide 1)是一种广泛存在于细胞浆中的异型寡聚蛋白,也是迄今为止真核细胞胞浆中发现的唯一伴侣素。目前认为大约15%的哺乳动物蛋白折叠需要CCT的参与,其中研究得最多的是肌动蛋白和微管蛋白。研究发现,CCT的异常会导致细胞骨架蛋白发生改变,甚至影响细胞骨架的形成与解聚。由此推测,一些细胞骨架相关疾病可能与CCT异常有关。     

The crystal structure of yeast CCT reveals intrinsic asymmetry of eukaryotic cytosolic chaperonins

The cytosolic chaperonin CCT is a 1‐MDa protein‐folding machine essential for eukaryotic life. The CCT interactome shows involvement in folding and assembly of a small range of proteins linked to essential cellular processes such as cytoskeleton assembly and cell‐cycle regulation. CCT has a classic chaperonin architecture, with two heterogeneous 8‐membered rings stacked back‐to‐back, enclosing a folding cavity. However, the mechanism by which CCT assists folding is distinct from other chaperonins, with no hydrophobic wall lining a potential Anfinsen cage, and a sequential rather than concerted ATP hydrolysis mechanism. We have solved the crystal structure of yeast CCT in complex with actin at 3.8 Å resolution, revealing the subunit organisation and the location of discrete patches of co‐evolving ‘signature residues’ that mediate specific interactions between CCT and its substrates. The intrinsic asymmetry is revealed by the structural individuality of the CCT subunits, which display unique configurations, substrate binding properties, ATP‐binding heterogeneity and subunit–subunit interactions. The location of the evolutionarily conserved N‐terminus of Cct5 on the outside of the barrel, confirmed by mutational studies, is unique to eukaryotic cytosolic chaperonins.     

The CCT chaperonin promotes activation of the anaphase-promoting complex through the generation of functional Cdc20

The WD repeat protein Cdc20 is essential for progression through mitosis because it is required to activate ubiquitin ligation by the anaphase-promoting complex (APC/C). Here we show in yeast that Cdc20 binds to the CCT chaperonin, which is known as a folding machine for actin and tubulin. The CCT is required for Cdc20's ability to bind and activate the APC/C. In vivo, CCT is essential for Cdc20-dependent cell cycle events such as sister chromatid separation and exit from mitosis. The chaperonin is also required for the function of the Cdc20-related protein Cdh1, which activates the APC/C during G1. We propose that folding of the Cdc20 family of APC/C activators is an essential and evolutionary conserved function of the CCT chaperonin.     

Caenorhabditis elegans chaperonin CCT/TRiC is required for actin and tubulin biogenesis and microvillus formation in intestinal epithelial cells

Intestinal epithelial cells have unique apical membrane structures, known as microvilli, that contain bundles of actin microfilaments. In this study, we report that Caenorhabditis elegans cytosolic chaperonin containing TCP-1 (CCT) is essential for proper formation of microvilli in intestinal cells. In intestinal cells of cct-5(RNAi) animals, a substantial amount of actin is lost from the apical area, forming large aggregates in the cytoplasm, and the apical membrane is deformed into abnormal, bubble-like structures. The length of the intestinal microvilli is decreased in these animals. However, the overall actin protein levels remain relatively unchanged when CCT is depleted. We also found that CCT depletion causes a reduction in the tubulin levels and disorganization of the microtubule network. In contrast, the stability and localization of intermediate filament protein IFB-2, which forms a dense filamentous network underneath the apical surface, appears to be superficially normal in CCT-deficient cells, suggesting substrate specificity of CCT in the folding of filamentous cytoskeletons in vivo. Our findings demonstrate physiological functions of CCT in epithelial cell morphogenesis using whole animals.     

A Two-step Mechanism for the Folding of Actin by the Yeast Cytosolic Chaperonin

Actin requires the chaperonin containing TCP1 (CCT), a hexadecameric ATPase essential for cell viability in eukaryotes, to fold to its native state. Following binding of unfolded actin to CCT, the cavity of the chaperone closes and actin is folded and released in an ATP-dependent folding cycle. In yeast, CCT forms a ternary complex with the phosducin-like protein PLP2p to fold actin, and together they can return nascent or chemically denatured actin to its native state in a pure in vitro folding assay. The complexity of the CCT-actin system makes the study of the actin folding mechanism technically challenging. We have established a novel spectroscopic assay through selectively labeling the C terminus of yeast actin with acrylodan and observe significant changes in the acrylodan fluorescence emission spectrum as actin is chemically unfolded and then refolded by the chaperonin. The variation in the polarity of the environment surrounding the fluorescent probe during the unfolding/folding processes has allowed us to monitor actin as it folds on CCT. The rate of actin folding at a range of temperatures and ATP concentrations has been determined for both wild type CCT and a mutant CCT, CCT4anc2, defective in folding actin in vivo. Binding of the non-hydrolysable ATP analog adenosine 5′-(β,γ-imino)triphosphate to the ternary complex leads to 3-fold faster release of actin from CCT following addition of ATP, suggesting a two-step folding process with a conformational change occurring upon closure of the cavity and a subsequent final folding step involving packing of the C terminus to the native-like state.     

Actin interacts with CCT via discrete binding sites: a binding transition-release model for CCT-mediated actin folding

The chaperones prefoldin and the cytosolic chaperonin CCT-containing TCP-1 (CCT) guide the cytoskeletal protein actin to its native conformation. Performing an alanine scan of actin, we identified discrete recognition determinants for CCT interaction. Interestingly, one of these is similar and functional in the non-homologous protein Cdc20, suggesting that some of the binding information in the CCT target proteins is shared. The information in actin for recognition by CCT and for folding is different, as all but one of the mutants in the recognition determinants are folding-competent. In addition, some other actin mutants remain CCT-arrested and are not released in a native conformation, whereas others do fold but remain bound to CAP. Kinetic experiments provide evidence that CCT-mediated folding of non-native actin occurs in at least two steps, in which initially the recognition determinant 245-249 contacts CCT and the other determinants interact at later stages. Actin mutants that are CCT-arrested demonstrate that some regions neighbouring the recognition determinants are involved in modulating the correct folding transitions of actin on CCT, or its release from this chaperonin. Further, we found that the ATP binding of actin is not a prerequisite for its release, and we suggest that CAP may be involved in charging the nucleotide. Based on the kinetics of CCT binding and folding of actin and actin mutants, we propose a multi-step recognition-transition-release model. This also implies that the currently accepted notion of CCT-mediated actin folding is probably more complex.     

Isolation and properties of two actin-binding domains in gelsolin

Gelsolin is a Ca2+-sensitive 90-kDa protein which regulates actin filament length. A molecular variant of gelsolin is present in plasma as a 93-kDa protein. Functional studies have shown that gelsolin contains two actin-binding sites which are distinct in that after Ca2+-mediated binding, removal of free Ca2+ releases actin from one site but not from the other. We have partially cleaved human plasma gelsolin with alpha-chymotrypsin and identified two distinct actin-binding domains. Peptides CT17 and CT15, which contain one of the actin-binding domains, bind to actin independently of Ca2+; peptides CT54 and CT47, which contain the other domain, bind to actin reversibly in response to changes in Ca2+ concentration. These peptides sequester actin monomers inhibiting polymerization. Unlike intact gelsolin, neither group of peptides nucleates actin assembly or forms stable filament end caps. CT17 and CT15 can however sever actin filaments. Amino acid sequence analyses place CT17 at the NH2 terminus of gelsolin and CT47 at the carboxyl-terminal two-thirds of gelsolin. Circular dichroism measurements show that Ca2+ induces an increase in the alpha-helical content of CT47. These studies provide a structural basis for understanding the interaction of gelsolin with actin and allow comparison with other Ca2+-dependent actin filament severing proteins.     

Analysis of the interaction between the eukaryotic chaperonin CCT and its substrates actin and tubulin

Two mechanisms have thus far been characterized for the assistance by chaperonins of the folding of other proteins. The first and best described is that of the prokaryotic chaperonin GroEL, which interacts with a large spectrum of proteins. GroEL uses a nonspecific mechanism by which any conformation of practically any unfolded polypeptide interacts with it through exposed, hydrophobic residues. ATP binding liberates the substrate in the GroEL cavity where it is given a chance to fold. A second mechanism has been described for the eukaryotic chaperonin CCT, which interacts mainly with the cytoskeletal proteins actin and tubulin. Cryoelectron microscopy and biochemical studies have revealed that both of these proteins interact with CCT in quasi-native, defined conformations. Here we have performed a detailed study of the docking of the actin and tubulin molecules extracted from their corresponding CCT:substrate complexes obtained from cryoelectron microscopy and image processing to localize certain regions in actin and tubulin that are involved in the interaction with CCT. These regions of actin and tubulin, which are not present in their prokaryotic counterparts FtsA and FtsZ, are involved in the polymerization of the two cytoskeletal proteins. These findings suggest coevolution of CCT with actin and tubulin in order to counteract the folding problems associated with the generation in these two cytoskeletal protein families of new domains involved in their polymerization.     

Quantitative actin folding reactions using yeast CCT purified via an internal tag in the CCT3/gamma subunit

The eukaryotic cytosolic chaperonin CCT is an essential ATP-dependent protein folding machine whose action is required for folding the cytoskeletal proteins actin and tubulin, and a small number of other substrates, including members of the WD40-propellor repeat-containing protein family. An efficient purification protocol for CCT from Saccharomyces cerevisiae has been developed. It uses the calmodulin binding peptide as an affinity tag in an internal loop in the apical domain of the CCT3 subunit, which is predicted to be located on the outside of the double-ring assembly. This purified yeast CCT was used for a novel quantitative actin-folding assay with human beta-actin or yeast ACT1p protein folding intermediates, Ac(I), pre-synthesised in an Escherichia coli translation system. The formation of native actin follows approximately a first-order reaction with a rate constant of about 0.03 min(-1). Yeast CCT catalyses the folding of yeast ACT1p and human beta-actin with nearly identical rate constants and yields. The results from this controlled CCT-actin folding assay are consistent with a model where CCT and Ac(I) are in a binding pre-equilibrium with a rate-limiting binding step, followed by a faster ATP-driven processing to native actin. In this pure in vitro system, the human beta-actin mutants, D244S and G150P, show impaired folding behaviour in the manner predicted by our sequence-specific recognition model for CCT-actin interaction.     

Individual subunits of the eukaryotic cytosolic chaperonin mediate interactions with binding sites located on subdomains of beta-actin

The chaperonin containing TCP-1 (CCT) of eukaryotic cytosol is composed of eight different subunit species that are proposed to have independent functions in folding its in vivo substrates, the actins and tubulins. CCT has been loaded with (35)S-beta-actin by in vitro translation in reticulocyte lysate and then subjected to immunoprecipitation with all eight anti-CCT subunit antibodies in mixed micelle buffers, conditions that disrupt CCT into its constituent monomers. Interactions between (35)S-beta-actin and isolated CCTalpha, CCTbeta, CCTepsilon, or CCTtheta subunits are observed, suggesting that polar and electrostatic interactions may mediate actin binding to these four CCT subunits. Additionally, a beta-actin peptide array was screened for CCT-binding sequences. Three regions rich in charged and polar amino acid residues, which map to the surface of native beta-actin, are implicated in interactions between actin and CCT. Several of these biochemical results are consistent with the recent cryo-electron microscopy three-dimensional structure of apo-CCT-alpha-actin, in which alpha-actin is bound by the apical domains of specific CCT subunits. A model is proposed in which actin interacts with several CCT subunits during its CCT-mediated folding cycle.     

Substantial CCT activity is required for cell cycle progression and cytoskeletal organization in mammalian cells

The chaperonin CCT hexadecamer is required for the folding of non-native actins and tubulins in eukaryotic cells. Among the consequences of greatly reducing CCT holocomplex levels in human cell lines by siRNA targeting are growth arrest and changes in cell morphology and motility. Less extensive reduction of CCT activity via microinjection of an inhibitory anti-CCT epsilon subunit monoclonal antibody, which alters the rates of substrate processing by CCT in vitro, causes a delay in cell cycle progression through G1/S phase in synchronized Swiss 3T3 cells. The degree of growth arrest strongly correlates with the extent of CCT depletion, indicating that full CCT activity is required for normal cell growth and division. Depletion of CCT does not affect actin polypeptide synthesis but causes a reduction in levels of native actin and perturbation of actin-based cell motility in BE cells. There are no large-scale effects on cytoplasmic protein synthesis or a general heat shock response during periods of low CCT activity.     

Yeast Phosducin-Like Protein 2 Acts as a Stimulatory Co-Factor for the Folding of Actin by the Chaperonin CCT via a Ternary Complex

The eukaryotic chaperonin-containing TCP-1 (CCT) folds the cytoskeletal protein actin. The folding mechanism of this 16-subunit, 1-MDa machine is poorly characterised due to the absence of quantitative in vitro assays. We identified phosducin-like protein 2, Plp2p (=PLP2), as an ATP-elutable binding partner of yeast CCT while establishing the CCT interactome. In a novel in vitro CCT-ACT1 folding assay that is functional under physiological conditions, PLP2 is a stimulatory co-factor. In a single ATP-driven cycle, PLP2-CCT-ACT1 complexes yield 30-fold more native actin than CCT-ACT1 complexes. PLP2 interacts directly with ACT1 through the C-terminus of its thioredoxin fold and the CCT-binding subdomain 4 of actin. The in vitro CCT-ACT1-PLP2 folding cycle of the preassembled complex takes 90 s at 30 °C, several times slower than the canonical chaperonin GroEL. The specific interactions between PLP2, CCT and ACT1 in the yeast-component in vitro system and the pronounced stimulatory effect of PLP2 on actin folding are consistent with in vivo genetic approaches demonstrating an essential and positive role for PLP2 in cellular processes involving actin in Saccharomyces cerevisiae. In mammalian systems, however, several members of the PLP family, including human PDCL3, the orthologue of PLP2, have been shown to be inhibitory toward CCT-mediated folding of actin in vivo and in vitro. Here, using a rabbit-reticulocyte-derived in vitro translation system, we found that inhibition of β-actin folding by PDCL3 can be relieved by exchanging its acidic C-terminal extension for that of PLP2. It seems that additional levels of regulatory control of CCT activity by this PLP have emerged in higher eukaryotes.     

A gelsolin-like protein from Papaver rhoeas pollen (PrABP80) stimulates calcium-regulated severing and depolymerization of actin filaments

The cytoskeleton is a key regulator of plant morphogenesis, sexual reproduction, and cellular responses to extracellular stimuli. During the self-incompatibility response of Papaver rhoeas L. (field poppy) pollen, the actin filament network is rapidly depolymerized by a flood of cytosolic free Ca2+ that results in cessation of tip growth and prevention of fertilization. Attempts to model this dramatic cytoskeletal response with known pollen actin-binding proteins (ABPs) revealed that the major G-actin-binding protein profilin can account for only a small percentage of the measured depolymerization. We have identified an 80-kDa, Ca(2+)-regulated ABP from poppy pollen (PrABP80) and characterized its biochemical properties in vitro. Sequence determination by mass spectrometry revealed that PrABP80 is related to gelsolin and villin. The molecular weight, lack of filament cross-linking activity, and a potent severing activity are all consistent with PrABP80 being a plant gelsolin. Kinetic analysis of actin assembly/disassembly reactions revealed that substoichiometric amounts of PrABP80 can nucleate actin polymerization from monomers, block the assembly of profilin-actin complex onto actin filament ends, and enhance profilin-mediated actin depolymerization. Fluorescence microscopy of individual actin filaments provided compelling, direct evidence for filament severing and confirmed the actin nucleation and barbed end capping properties. This is the first direct evidence for a plant gelsolin and the first example of efficient severing by a plant ABP. We propose that PrABP80 functions at the center of the self-incompatibility response by creating new filament pointed ends for disassembly and by blocking barbed ends from profilin-actin assembly.     

The interaction network of the chaperonin CCT

The eukaryotic cytosolic chaperonin containing TCP-1 (CCT) has an important function in maintaining cellular homoeostasis by assisting the folding of many proteins, including the cytoskeletal components actin and tubulin. Yet the nature of the proteins and cellular pathways dependent on CCT function has not been established globally. Here, we use proteomic and genomic approaches to define CCT interaction networks involving 136 proteins/genes that include links to the nuclear pore complex, chromatin remodelling, and protein degradation. Our study also identifies a third eukaryotic cytoskeletal system connected with CCT: the septin ring complex, which is essential for cytokinesis. CCT interactions with septins are ATP dependent, and disrupting the function of the chaperonin in yeast leads to loss of CCT-septin interaction and aberrant septin ring assembly. Our results therefore provide a rich framework for understanding the function of CCT in several essential cellular processes, including epigenetics and cell division.