RESUMEN

Actin is dependent on the type-II chaperonin CCT (chaperonin containing TCP-1) to reach its native state. In vitro, yeast CCT folds yeast and also mammalian cytoplasmic (beta/gamma) actins but is now found to be incapable of folding mammalian skeletal muscle alpha-actin. Arrest of alpha-actin on yeast CCT at a folding cycle intermediate has been observed by electron microscopy. This discovery explains previous observations in vivo that yeast mutants expressing only the muscle actin gene are non-viable. Mutational analysis identified a single specific alpha-actin residue, Asn-297, that confers this species/isoform folding specificity. The implications of this incompatibility for chaperonin mechanism and actin-CCT co-evolution are discussed.

Asunto(s)

Actinas/química , Actinas/metabolismo , Aminoácidos/metabolismo , Chaperoninas/química , Chaperoninas/metabolismo , Actinas/genética , Actinas/aislamiento & purificación , Actinas/ultraestructura , Secuencia de Aminoácidos , Animales , Asparagina/metabolismo , Chaperonina con TCP-1 , Chaperoninas/genética , Chaperoninas/aislamiento & purificación , Chaperoninas/ultraestructura , Escherichia coli/genética , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Músculo Esquelético/química , Mutación , Conformación Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Conejos , Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de Aminoácido , Termodinámica

RESUMEN

We present a platform for the spatially selective sampling of the plasma membrane of single cells. Optically trapped lipid-coated oil droplets (smart droplet microtools, SDMs), typically 0.5-5 microm in size, composed of a hexadecane hydrocarbon core and fusogenic lipid outer coating (mixture of 1,2-dioleoyl-phosphatidylethanolamine and 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine) were brought into controlled contact with target colon cancer cells leading to the formation of connecting membrane tethers. Material transfer from the cell to the SDM across the membrane tether was monitored by tracking membrane-localized enhanced green fluorescent protein.

Asunto(s)

Membrana Celular/química , Separación Celular , Proteínas de la Membrana/análisis , Proteómica/métodos , Compartimento Celular , Fraccionamiento Celular , Células Cultivadas , Técnicas Citológicas/instrumentación , Emulsiones , Humanos , Lípidos , Fusión de Membrana , Microscopía Fluorescente , Pinzas Ópticas , Óptica y Fotónica , Proteómica/instrumentación

RESUMEN

The human tumor suppressor gene ataxia telangiectasia mutated (ATM) encodes a 3056 amino-acid protein kinase that regulates cell cycle checkpoints. ATM is defective in the neurodegenerative and cancer predisposition syndrome ataxia-telangiectasia. ATM protein kinase is activated by DNA damage and responds by phosphorylating downstream effectors involved in cell cycle arrest and DNA repair, such as p53, MDM2, CHEK2, BRCA1 and H2AX. ATM is probably a component of, or in close proximity to, the double-stranded DNA break-sensing machinery. We have observed purified human ATM protein, ATM-DNA and ATM-DNA-avidin bound complexes by single-particle electron microscopy and obtained three-dimensional reconstructions which show that ATM is composed of two main domains comprising a head and an arm. DNA binding to ATM induces a large conformational movement of the arm-like domain. Taken together, these three structures suggest that ATM is capable of interacting with DNA, using its arm to clamp around the double helix.

Asunto(s)

ADN/metabolismo , Proteínas Serina-Treonina Quinasas/química , Proteínas de la Ataxia Telangiectasia Mutada , Avidina/metabolismo , Proteínas de Ciclo Celular , ADN/química , Daño del ADN , Proteínas de Unión al ADN , Humanos , Procesamiento de Imagen Asistido por Computador , Sustancias Macromoleculares , Microscopía Electrónica , Modelos Moleculares , Conformación de Ácido Nucleico , Unión Proteica , Conformación Proteica , Mapeo de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/ultraestructura , Estructura Terciaria de Proteína , Proteínas Supresoras de Tumor

Asunto(s)

Antibacterianos/uso terapéutico , Arginina/genética , Codón sin Sentido/genética , Proteínas del Ojo , Gentamicinas/uso terapéutico , Proteínas/genética , Retinitis Pigmentosa/tratamiento farmacológico , Retinitis Pigmentosa/genética , Animales , Células CHO , Línea Celular , Cricetinae , Proteínas de Unión al GTP , Humanos , Péptidos y Proteínas de Señalización Intracelular , Masculino , Proteínas de la Membrana

RESUMEN

The three-dimensional reconstruction of apo-CCT-alpha-actin by cryoelectron microscopy shows that actin binds either the CCTbeta-CCTdelta or the CCTepsilon-CCTdelta subunit pairs of the chaperonin in an open and apparently quasi-native conformation. The CCT-binding sites are seen located at the tips of the two arms of actin and these same regions of actin have been implicated in CCT binding through beta-actin peptide-array screening. Three main CCT binding regions exist: actin Sites I, II, and III, which are composed of loops that are surface-exposed in native actin. Sixty-eight amino acid residues on beta-actin have been screened by mutagenesis for effects on CCT interaction in quantitative in vitro translation assays in rabbit reticulocyte lysate. Actin seems to be folding cooperatively on chaperonin, since certain mutants discriminate CCT binding from processing. Actin Site II, located at the tip of actin subdomain 4, is the major determinant for CCT binding. Site II is composed of two anti-parallel extended beta-strands, with F200-T203 and D244 contributing substantially to the binding site. The substrate recognition chemistry of CCT thus seems different from that of Group I chaperonins and probably reflects the fact that it needs to be highly specific to enable capture and folding of the actins and tubulins.

Asunto(s)

Actinas/química , Chaperoninas/metabolismo , Proteínas del Citoesqueleto , Proteínas de Drosophila , Proteínas de Microfilamentos , Mutagénesis Sitio-Dirigida , Pliegue de Proteína , Actinas/genética , Actinas/metabolismo , Animales , Sitios de Unión , Proteínas de Ciclo Celular , Sistema Libre de Células , Chaperonina con TCP-1 , Chaperoninas/química , Citosol , Células Eucariotas , Humanos , Cinética , Ratones , Unión Proteica , Estructura Terciaria de Proteína , Especificidad por Sustrato

RESUMEN

The 30-A cryo-EM-derived structure of apo-CCT-alpha-actin shows actin opened up across its nucleotide-binding cleft and binding to either of two CCT subunit pairs, CCTbeta-CCTdelta or CCTepsilon-CCTdelta, in a similar 1:4 arrangement. The two main duplicated domains of native actin are linked twice, topologically, by the connecting residues, Q137-S145 and P333-S338, and are tightly held together by hydrogen bonding with bound adenine nucleotide. We carried out a mutational screen to find residues in actin that might be involved in the huge rotations observed in the CCT-bound folding intermediate. When two evolutionarily highly conserved glycine residues of beta-actin, G146 and G150, were changed to proline, both mutant actin proteins were poorly processed by CCT in in vitro translation assays; they become arrested on CCT. A three-dimensional reconstruction of the substrate-bound ring of the apo-CCT-beta-actin complex shows that beta-actin G150P is not able to bind across the chaperonin cavity to interact with the CCTdelta subunit. beta-actin G150P seems tightly packed and apparently bound only to the CCTbeta and CCTepsilon subunits, which further indicates that these CCT subunits drive the interaction between CCT and actin. Hinge opening seems to be critical for actin folding, and we suggest that residues G146 and G150 are important components of the hinge around which the rigid subdomains, presumably already present in early actin folding intermediates, rotate during CCT-assisted folding.

Asunto(s)

Actinas/química , Péptidos y Proteínas de Señalización Intracelular , Proteínas Asociadas a Microtúbulos , Proteínas Nucleares/química , Mutación Puntual , Pliegue de Proteína , Actinas/genética , Actinas/metabolismo , Secuencia de Aminoácidos , Citosol , Humanos , Imagenología Tridimensional , Cinética , Microscopía Electrónica , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Ubiquitina-Proteína Ligasas , Región del Complejo T del Genoma

RESUMEN

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.

Asunto(s)

Actinas/metabolismo , Proteínas del Citoesqueleto , Péptidos y Proteínas de Señalización Intracelular , Proteínas Asociadas a Microtúbulos , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Tubulina (Proteína)/metabolismo , Actinas/química , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/química , Sitios de Unión , Bovinos , Microscopía por Crioelectrón , Células Eucariotas , Humanos , Imagenología Tridimensional , Datos de Secuencia Molecular , Unión Proteica , Pliegue de Proteína , Alineación de Secuencia , Especificidad por Sustrato , Tubulina (Proteína)/química , Ubiquitina-Proteína Ligasas , Región del Complejo T del Genoma

RESUMEN

Folding to completion of actin and tubulin in the eukaryotic cytosol requires their interaction with cytosolic chaperonin CCT [chaperonin containing tailless complex polypeptide 1 (TCP-1)]. Three-dimensional reconstructions of nucleotide-free CCT complexed to either actin or tubulin show that CCT stabilizes both cytoskeletal proteins in open and quasi-folded conformations mediated through interactions that are both subunit specific and geometry dependent. Here we find that upon ATP binding, mimicked by the non-hydrolysable analog AMP-PNP (5'-adenylyl-imido-diphosphate), to both CCT-alpha-actin and CCT- beta-tubulin complexes, the chaperonin component undergoes concerted movements of the apical domains, resulting in the cavity being closed off by the helical protrusions of the eight apical domains. However, in contrast to the GroE system, generation of this closed state does not induce the release of the substrate into the chaperonin cavity, and both cytoskeletal proteins remain bound to the chaperonin apical domains. Docking of the AMP-PNP-CCT-bound conformations of alpha-actin and beta-tubulin to their respective native atomic structures suggests that both proteins have progressed towards their native states.

Asunto(s)

Actinas/química , Chaperoninas/química , Pliegue de Proteína , Tubulina (Proteína)/química , Células 3T3 , Adenilil Imidodifosfato/química , Sitio Alostérico , Animales , Chaperonina con TCP-1 , Células Eucariotas , Humanos , Imagenología Tridimensional , Ratones , Microscopía Inmunoelectrónica/métodos , Modelos Moleculares , Conformación Proteica , Ratas

RESUMEN

Initial rates of ATP hydrolysis by the chaperonin containing TCP-1 (CCT) from bovine testis were measured as a function of ATP concentration. Two allosteric transitions are observed: one at relatively low concentrations of ATP (<100 microM) and the second at higher concentrations of ATP. The data suggest that CCT has positive intra-ring cooperativity and negative inter-ring cooperativity in ATP hydrolysis, with respect to ATP, as previously observed in the case of GroEL. It is shown that the relatively weak positive intra-ring cooperativity found in the case of CCT may be due to heterogeneity in its subunit composition. Our results suggest that nested allosteric behavior may be common to chaperone double-ring systems.

Asunto(s)

Chaperoninas/química , Citoplasma/química , Testículo/química , Adenosina Trifosfato/metabolismo , Sitio Alostérico , Animales , Bovinos , Chaperonina 60/química , Chaperonina con TCP-1 , Dimerización , Hidrólisis , Cinética , Masculino , Unión Proteica

RESUMEN

Three-dimensional reconstruction from cryoelectron micrographs of the eukaryotic cytosolic chaperonin CCT complexed to tubulin shows that CCT interacts with tubulin (both the alpha and beta isoforms) using five specific CCT subunits. The CCT-tubulin interaction has a different geometry to the CCT-actin interaction, and a mixture of shared and unique CCT subunits is used in binding the two substrates. Docking of the atomic structures of both actin and tubulin to their CCT-bound conformation suggests a common mode of chaperonin-substrate interaction. CCT stabilizes quasi-native structures in both proteins that are open through their domain-connecting hinge regions, suggesting a novel mechanism and function of CCT in assisted protein folding.

Asunto(s)

Actinas/química , Actinas/ultraestructura , Chaperoninas/química , Chaperoninas/ultraestructura , Tubulina (Proteína)/química , Tubulina (Proteína)/ultraestructura , Actinas/genética , Animales , Apoproteínas/química , Apoproteínas/genética , Apoproteínas/ultraestructura , Sitios de Unión , Chaperonina con TCP-1 , Chaperoninas/genética , Microscopía por Crioelectrón , Estabilidad de Medicamentos , Evolución Molecular , Humanos , Procesamiento de Imagen Asistido por Computador , Técnicas In Vitro , Conformación Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/ultraestructura , Tubulina (Proteína)/genética

RESUMEN

The actins and tubulins are the obligate substrates in vivo of the chaperonin-containing TCP-1 (CCT). The precise elements of recognition between the chaperonin and its substrates remain largely unknown. We have used a solid phase peptide binding assay to screen the human alpha, beta and gamma-tubulin sequences for CCT recognition. Multiple regions seem to be implicated in interactions between tubulins and CCT. These potential CCT-binding sites are highly dispersed throughout the primary sequences of the human tubulins. In addition, using site-directed mutagenesis we assessed the contribution of the selected residues in the C-terminal domain of beta-tubulin to CCT binding. Various hot spots have been identified even though, in each case, their replacement by alanine does not reduce dramatically the total affinity of beta-tubulin for CCT. The CCT-binding information in the tubulins is probably confined to multiple specific regions each having weak or moderate affinity for CCT apical domains. The main binding region seems to be located between residues 263 and 384, but there are no single amino acid residues in this region, which make large contributions to the binding energy, although we have detected a minor contribution by F377. These biochemical results are understandable in the context of our recent structural analysis of CCT-tubulin complexes by cryo-electron microscopy and image reconstruction, which shows that, in one stage of an in vitro binding reaction between apo-CCT and tubulin diluted from guanidinium chloride, ten major, stable contacts between tubulin and CCT are involved. Therefore, specificity is achieved through the co-operation of many specific, albeit weak, interactions.

Asunto(s)

Chaperoninas/metabolismo , Citosol/metabolismo , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Chaperoninas/clasificación , Citosol/química , ADN Complementario/genética , Humanos , Masculino , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación/genética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Especificidad por Sustrato , Testículo/citología , Termodinámica , Tubulina (Proteína)/genética

RESUMEN

The X-linked retinitis pigmentosa (XLRP) gene, RP2, codes for a novel 350 amino acid protein of unknown function. We have identified putative sites for N-terminal acyl modification by myristoylation and palmitoylation in the RP2 protein. The RP2 protein is expressed ubiquitously in human tissues at relatively low levels (0.01% of total protein) and has a predominantly plasma membrane localization in cultured cells, as would be expected if the protein was subject to dual N-terminal acylation. Furthermore, mutagenesis of residues potentially required for N-terminal acylation prevents targeting of RP2 to the plasma membrane and the N-terminal 15 amino acids of the protein appear to be sufficient for this targeting. Our data suggest that the protein is dually acylated and that the palmitoyl moiety is responsible for targeting of the myristoylated protein from intracellular membranes to the plasma membrane. The effect of two mutations, which have been reported as causes of XLRP, R118H and DeltaS6, were investigated. The R118H mutation does not affect the normal plasma membrane localization of RP2; in contrast, the DeltaS6 mutation interferes with the targeting of the protein to the plasma membrane. Therefore, the DeltaS6 mutation may cause XLRP because it prevents normal amounts of RP2 reaching the correct cellular locale, whereas the R118H mutation is in a region of the protein that is vital for another aspect of RP2 function in the retina.

Asunto(s)

Proteínas del Ojo , Proteínas de la Membrana/genética , Proteínas/genética , Retinitis Pigmentosa/genética , Cromosoma X , Acilación , Secuencia de Aminoácidos , Animales , Western Blotting , Células CHO , Membrana Celular/metabolismo , Cricetinae , Técnica del Anticuerpo Fluorescente , Proteínas de Unión al GTP , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana/metabolismo , Ratones , Microscopía Confocal , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Proteínas/metabolismo , Ratas , Retinitis Pigmentosa/metabolismo , Eliminación de Secuencia , Fracciones Subcelulares/metabolismo , Células Tumorales Cultivadas

Asunto(s)

Chaperoninas/historia , Ratones/genética , Animales , Chaperonina con TCP-1 , Chaperoninas/genética , Haplotipos , Historia del Siglo XX , Pliegue de Proteína , Estructura Terciaria de Proteína , Reino Unido

RESUMEN

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.

Asunto(s)

Actinas/metabolismo , Chaperoninas/metabolismo , Citosol/metabolismo , Actinas/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Chaperonina con TCP-1 , Chaperoninas/química , Chaperoninas/genética , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis , Conformación Proteica

RESUMEN

The eukaryotic chaperonin containing T-complex polypeptide 1 (CCT) is required in vivo for the production of native actin and tubulin. It is a 900-kDa oligomer formed from two back-to-back rings, each containing eight different subunits surrounding a central cavity in which interactions with substrates are thought to occur. Here, we show that a monoclonal antibody recognizing the C terminus of the CCTalpha subunit can bind inside, and partially occlude, both cavities of apo-CCT. Rabbit reticulocyte lysate was programmed to synthesize beta-actin and alpha-tubulin in the presence and absence of anti-CCTalpha antibody. The binding of the antibody inside the cavity and its occupancy of a large part of it does not prevent the folding of beta-actin and alpha-tubulin by CCT, despite the fact that all the CCT in the in vitro translation reactions was continuously bound by two antibody molecules. Furthermore, no differences in the protease susceptibility of actin bound to CCT in the presence and absence of the monoclonal antibody were detected, indicating that the antibody molecules do not perturb the conformation of actin folding intermediates substantially. These data indicate that complete sequestration of substrate by CCT may not be required for productive folding, suggesting that there are differences in its folding mechanism compared with the Group I chaperonins.

Asunto(s)

Actinas/metabolismo , Chaperoninas/antagonistas & inhibidores , Pliegue de Proteína , Tubulina (Proteína)/metabolismo , Animales , Chaperonina con TCP-1 , Chaperoninas/química , Microscopía Electrónica , Conformación Proteica , Conejos

RESUMEN

Chaperonins assist the folding of other proteins. Type II chaperonins, such as chaperonin containing TCP-1(CCT), are found in archaea and in the eukaryotic cytosol. They are hexadecameric or nonadecameric oligomers composed of one to eight different polypeptides. Whereas type I chaperonins like GroEL are promiscuous, assisting in the folding of many other proteins, only a small number of proteins, mainly actin and tubulin, have been described as natural substrates of CCT. This specificity may be related to the divergence of the eight CCT subunits. Here we have obtained a three-dimensional reconstruction of the complex between CCT and alpha-actin by cryo-electron microscopy and image processing. This shows that alpha-actin interacts with the apical domains of either of two CCT subunits. Immunolabelling of CCT-substrate complexes with antibodies against two specific CCT subunits showed that actin binds to CCT using two specific and distinct interactions: the small domain of actin binds to CCTdelta and the large domain to CCTbeta or CCTepsilon (both in position 1,4 with respect to delta). These results indicate that the binding of actin to CCT is both subunit-specific and geometry-dependent. Thus, the substrate recognition mechanism of eukaryotic CCT may differ from that of prokaryotic GroEL.

Asunto(s)

Actinas/metabolismo , Chaperoninas/metabolismo , Actinas/química , Actinas/genética , Animales , Sitios de Unión , Chaperonina con TCP-1 , Clonación Molecular , Escherichia coli , Humanos , Modelos Moleculares , Conformación Proteica , Ratas , Proteínas Recombinantes de Fusión/metabolismo

RESUMEN

The type II chaperonin CCT (chaperonin containing Tcp-1) of eukaryotic cytosol is a heteromeric 16-mer particle composed of eight different subunits. Three-dimensional reconstructions of apo-CCT and ATP-CCT have been obtained at 28 A resolution by cryo-electron microscopy. Binding of ATP generates an asymmetric particle; one ring has a slightly different conformation from the apo-CCT ring, while the other has undergone substantial movements in the apical domains. Upon ATP binding the apical domains rotate and point towards the cylinder axis, so that the helical protrusions present at their tips could act as a lid closing the ring cavity.

Asunto(s)

Adenosina Trifosfato/química , Proteínas Arqueales , Proteínas de Choque Térmico/química , Chaperonas Moleculares/química , Conformación Proteica , Pliegue de Proteína , Animales , Microscopía por Crioelectrón , Masculino , Ratones , Unión Proteica , Testículo/química , Thermoplasma/química

Asunto(s)

Proteínas Quinasas/genética , Razón de Masculinidad , Espermatozoides/fisiología , Animales , Femenino , Haplotipos , Masculino , Meiosis , Ratones , Proteínas Quinasas/fisiología

RESUMEN

The chaperonin-containing TCP-1 (CCT) assists in the folding of actins and tubulins in eukaryotic cells. CCT is composed of 8 subunit species encoded by separate genes. CCT purifies as a single hetero-oligomeric protein complex of 950 kDa through multiple chromatographic and antibody affinity procedures. The CCT 16-mer contains 7 polypeptide species in equimolar amounts (CCTalpha, beta, gamma, delta, epsilon, zeta, eta), together with another subunit (CCTtheta) which is around half-molar. Here we show, by in vitro translation of CCT subunit mRNAs in rabbit reticulocyte lysate, that none of the CCT subunit proteins are themselves folded by CCT. However, the newly translated CCT subunits can incorporate into the endogenous CCT complex present in the lysate via a mechanism involving a nucleotide-dependent disassembly reaction to produce single-rings and then a reassembly reaction whereby free CCT subunits assemble onto these single-rings. This cycling behaviour is an inherent property of the CCT chaperonin complex and provides a powerful method for introducing single amino acid residue changes into this 8578 residue protein complex.

Asunto(s)

Chaperoninas/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Chaperonina con TCP-1 , Chaperoninas/genética , ADN , Cinética , Ratones , Datos de Secuencia Molecular , Mutagénesis , Pliegue de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Conejos , Reticulocitos/metabolismo

RESUMEN

The chaperonin-containing TCP-1 complex (CCT) is a heteromeric particle composed of eight different subunits arranged in two back-to-back 8-fold pseudo-symmetric rings. The structural and functional implications of nucleotide binding to the CCT complex was addressed by electron microscopy and image processing. Whereas ADP binding to CCT does not reveal major conformational differences when compared with nucleotide-free CCT, ATP binding induces large conformational changes in the apical and equatorial domains, shifting the latter domains up to 40 degrees (with respect to the inter-ring plane) compared with 10 degrees for nucleotide-free CCT or ADP-CCT. This equatorial ATP-induced shift has no counterpart in GroEL, its prokaryotic homologue, which suggests differences in the folding mechanism for CCT.

Asunto(s)

Adenosina Trifosfato/metabolismo , Chaperoninas/metabolismo , Animales , Chaperonina con TCP-1 , Chaperoninas/química , Ratones , Microscopía Electrónica , Conformación Proteica