Identification and functional analysis of chaperonin 10, the groES homolog from yeast mitochondria.
ABSTRACT: Chaperonin 60 (cpn60) and chaperonin 10 (cpn10) constitute the chaperonin system in prokaryotes, mitochondria, and chloroplasts. In Escherichia coli, these two chaperonins are also termed groEL and groES. We have used a functional assay to identify the groES homolog cpn10 in yeast mitochondria. When dimeric ribulose-1,5-bisphosphate carboxylase (Rubisco) is denatured and allowed to bind to yeast cpn60, subsequent refolding of Rubisco is strictly dependent upon yeast cpn10. The heterologous combination of cpn60 from E. coli plus yeast cpn10 is also functional. In contrast, yeast cpn60 plus E. coli cpn10 do not support refolding of Rubisco. In the presence of MgATP, yeast cpn60 and yeast cpn10 form a stable complex that can be isolated by gel filtration and that facilitates refolding of denatured Rubisco. Although the potassium-dependent ATPase activity of E. coli cpn60 can be inhibited by cpn10 from either E. coli or yeast, neither of these cpn10s inhibits the ATPase activity of yeast cpn60. Amino acid sequencing of yeast cpn10 reveals substantial similarity to the corresponding cpn10 proteins from rat mitochondria and prokaryotes.
Project description:Chloroplasts of higher plants contain a nuclear-encoded protein that is a functional homolog of the Escherichia coli chaperonin 10 (cpn10; also known as groES). In pea (Pisum sativum), chloroplast cpn10 was identified by its ability to (i) assist bacterial chaperonin 60 (cpn60; also known as groEL) in the ATP-dependent refolding of chemically denatured ribulose-1,5-bisphosphate carboxylase and (ii) form a stable complex with bacterial cpn60 in the presence of Mg.ATP. The subunit size of the pea protein is approximately 24 kDa--about twice the size of bacterial cpn10. A cDNA encoding a spinach (Spinacea oleracea) chloroplast cpn10 was isolated, sequenced, and expressed in vitro. The spinach protein is synthesized as a higher molecular mass precursor and has a typical chloroplast transit peptide. Surprisingly, however, attached to the transit peptide is a single protein, comprised of two distinct cpn10 molecules in tandem. Moreover, both halves of this "double" cpn10 are highly conserved at a number of residues that are present in all cpn10s that have been examined. Upon import into chloroplasts the spinach cpn10 precursor is processed to its mature form of approximately 24 kDa. N-terminal amino acid sequence analysis reveals that the mature pea and spinach cpn10 are identical at 13 of 21 residues.
Project description:In vitro refolding of pig mitochondrial malate dehydrogenase is investigated in the presence of Escherichia coli chaperonins cpn60 (groEL) and cpn10 (groES). When the enzyme is initially denatured with 3 M guanidinium chloride, chaperonin-assisted refolding is 100% efficient. C.d. spectroscopy reveals that malate dehydrogenase is almost unfolded in 3 M guanidinium chloride, suggesting that a state with little or no residual secondary structure is the optimal 'substrate' for chaperonin-assisted refolding. Malate dehydrogenase denatured to more highly structured states proves to refold less efficiently with chaperonin assistance. The enzyme is shown not to aggregate under the refolding conditions, so that losses in refolding efficiency result from irreversible misfolding. Evidence is advanced to suggest that the chaperonins are unable to rescue irreversibly misfolded malate dehydrogenase. A novel use is made of 100 K Centricon concentrators to study the binding of [14C]acetyl-labelled malate dehydrogenase to groEL by an ultrafiltration binding assay. Analysis of the data by Scatchard plot shows that acetyl-malate dehydrogenase, which has previously been extensively unfolded with guanidinium chloride, binds to groEL at a specific binding site(s). At saturation, one acetyl-malate dehydrogenase homodimer (two polypeptides) is shown to bind to each groEL homooligomer with a binding constant of approx. 10 nM.
Project description:In vitro refolding of pig mitochondrial malate dehydrogenase is investigated in the presence and absence of Escherichia coli chaperonins cpn60 (groEL) and cpn10 (groES). The refolded yields of active malate dehydrogenase are increased almost 3-fold in the presence of groEL, groES, Mg2+/ATP and K+ ions. Chaperonin-assisted refolding of malate dehydrogenase does not have an absolute requirement for K+ ions but Mg2+/ATP is obligatory. When ATP is replaced by other nucleoside triphosphates, or by non-hydrolysable ATP analogues, assisted refolding is prevented. Optimal chaperonin-assisted refolding requires both groEL and groES homo-oligomers in molar excess over malate dehydrogenase. Kinetic analysis shows that the chaperonins do not catalyse the refolding of malate dehydrogenase but increase the flux of unfolded enzyme through the productive refolding pathway without altering and/or accelerating that pathway. Although not acting as refolding catalysts, the chaperonins are able to assist at least six consecutive cycles of malate dehydrogenase refolding.
Project description:The refolding of lactate dehydrogenase fully unfolded in 4 M guanidinium chloride was initiated by dilution into assay buffer, and the emergence of active enzyme was recorded. This was performed in the presence of the following chaperonin complexes in the refolding medium: chaperonin-60 (cpn60), cpn60-MgATP, cpn60-Mgp[NH]ppA, cpn60-MgADP in both the presence and absence of chaperonin-10 (cpn10). For each nucleotide-chaperonin complex studied, the effect of nucleotide concentration was measured. Dissociation constants (Kd) for unfolded LDH bound to the various chaperonin complexes were derived directly from the ability of the complexes to retard the folding of the enzyme. Dissociation constants for the different complexes were found to be in the order: cpn60 < cpn60-MgADP-cpn10 (formed at low [MgADP]) < cpn60-MgADP < cpn60-MgADP-cpn10 < cpn60-Mgp[NH]ppA < cpn60-Mgp[NH]ppA-cpn10 < cpn60-MgATP < cpn60-MgATP-cpn10; i.e. the tightest complex is with cpn60 and the weakest with cpn60-MgATP-cpn10. Only when MgATP is the nucleotide do we see the yield of native enzyme increased on the time scale of 1 h. The results provide estimates of the change in binding energy between the chaperonin and a substrate protein through the cycle of MgATP binding, hydrolysis and dissociation.
Project description:Co-chaperonin protein 10 (cpn10, GroES in Escherichia coli) is a ring-shaped heptameric protein that facilitates substrate folding when in complex with cpn60 (GroEL in E. coli). The cpn10 from the hyperthermophilic, ancient bacterium Aquifex aeolicus (Aacpn10) has a 25-residue C-terminal extension in each monomer not found in any other cpn10 protein. Earlier in vitro work has shown that this tail is not needed for heptamer assembly or protein function. Without the tail, however, the heptamers (Aacpn10del-25) readily aggregate into fibrillar stacked rings. To explain this phenomenon, we performed binding experiments with a peptide construct of the tail to establish its specificity for Aacpn10del-25 and used cryo-electron microscopy to determine the three-dimensional (3D) structure of the GroEL-Aacpn10-ADP complex at an 8-A resolution. We found that the GroEL-Aacpn10 structure is similar to the GroEL-GroES structure at this resolution, suggesting that Aacpn10 has molecular interactions with cpn60 similar to other cpn10s. The cryo-electron microscopy density map does not directly reveal the density of the Aacpn10 25-residue tail. However, the 3D statistical variance coefficient map computed from multiple 3D reconstructions with randomly selected particle images suggests that the tail is located at the Aacpn10 monomer-monomer interface and extends toward the cis-ring apical domain of GroEL. The tail at this location does not block the formation of a functional co-chaperonin/chaperonin complex but limits self-aggregation into linear fibrils at high temperatures. In addition, the 3D variance coefficient map identifies several regions inside the GroEL-Aacpn10 complex that have flexible conformations. This observation is in full agreement with the structural properties of an effective chaperonin.
Project description:The crystal structure of Mycobacterium tuberculosis chaperonin 10 (cpn10(Mt)) has been determined to a resolution of 2.8 A. Two dome-shaped cpn10(Mt) heptamers complex through loops at their bases to form a tetradecamer with 72 symmetry and a spherical cage-like structure. The hollow interior enclosed by the tetradecamer is lined with hydrophilic residues and has dimensions of 30 A perpendicular to and 60 A along the sevenfold axis. Tetradecameric cpn10(Mt) has also been observed in solution by dynamic light scattering. Through its base loop sequence cpn10(Mt) is known to be the agent in the bacterium responsible for bone resorption and for the contribution towards its strong T-cell immunogenicity. Superimposition of the cpn10(Mt) sequences 26 to 32 and 66 to 72 and E. coli GroES 25 to 31 associated with bone resorption activity shows them to have similar conformations and structural features, suggesting that there may be a common receptor for the bone resorption sequences. The base loops of cpn10s in general also attach to the corresponding chaperonin 60 (cpn60) to enclose unfolded protein and to facilitate its correct folding in vivo. Electron density corresponding to a partially disordered protein subunit appears encapsulated within the interior dome cavity of each heptamer. This suggests that the binding of substrates to cpn10 is possible in the absence of cpn60.
Project description:Type I chaperonins (cpn60/Hsp60) are essential proteins that mediate the folding of proteins in bacteria, chloroplast and mitochondria. Despite the high sequence homology among chaperonins, the mitochondrial chaperonin system has developed unique properties that distinguish it from the widely-studied bacterial system (GroEL and GroES). The most relevant difference to this study is that mitochondrial chaperonins are able to refold denatured proteins only with the assistance of the mitochondrial co-chaperonin. This is in contrast to the bacterial chaperonin, which is able to function with the help of co-chaperonin from any source. The goal of our work was to determine structural elements that govern the specificity between chaperonin and co-chaperonin pairs using mitochondrial Hsp60 as model system. We used a mutagenesis approach to obtain human mitochondrial Hsp60 mutants that are able to function with the bacterial co-chaperonin, GroES. We isolated two mutants, a single mutant (E321K) and a double mutant (R264K/E358K) that, together with GroES, were able to rescue an E. coli strain, in which the endogenous chaperonin system was silenced. Although the mutations are located in the apical domain of the chaperonin, where the interaction with co-chaperonin takes place, none of the residues are located in positions that are directly responsible for co-chaperonin binding. Moreover, while both mutants were able to function with GroES, they showed distinct functional and structural properties. Our results indicate that the phenotype of the E321K mutant is caused mainly by a profound increase in the binding affinity to all co-chaperonins, while the phenotype of R264K/E358K is caused by a slight increase in affinity toward co-chaperonins that is accompanied by an alteration in the allosteric signal transmitted upon nucleotide binding. The latter changes lead to a great increase in affinity for GroES, with only a minor increase in affinity toward the mammalian mitochondrial co-chaperonin.
Project description:The chloroplast chaperonin system of plants and green algae is a curiosity as both the chaperonin cage and its lid are encoded by multiple genes, in contrast to the single genes encoding the two components of the bacterial and mitochondrial systems. In the green alga Chlamydomonas reinhardtii (Cr), three genes encode chaperonin cofactors, with cpn10 encoding a single ?10-kDa domain and cpn20 and cpn23 encoding tandem cpn10 domains. Here, we characterized the functional interaction of these proteins with the Escherichia coli chaperonin, GroEL, which normally cooperates with GroES, a heptamer of ?10-kDa subunits. The C. reinhardtii cofactor proteins alone were all unable to assist GroEL-mediated refolding of bacterial ribulose-bisphosphate carboxylase/oxygenase but gained this ability when CrCpn20 and/or CrCpn23 was combined with CrCpn10. Native mass spectrometry indicated the formation of hetero-oligomeric species, consisting of seven ?10-kDa domains. The cofactor "heptamers" interacted with GroEL and encapsulated substrate protein in a nucleotide-dependent manner. Different hetero-oligomer arrangements, generated by constructing cofactor concatamers, indicated a preferential heptamer configuration for the functional CrCpn10-CrCpn23 complex. Formation of heptamer Cpn10/Cpn20 hetero-oligomers was also observed with the Arabidopsis thaliana (At) cofactors, which functioned with the chloroplast chaperonin, AtCpn60?(7)?(7). It appears that hetero-oligomer formation occurs more generally for chloroplast chaperonin cofactors, perhaps adapting the chaperonin system for the folding of specific client proteins.
Project description:Chaperonin GroEL (Cpn60) requires cofactor GroES (Cpn10) for protein refolding in bacteria that possess single <i>groEL</i> and <i>groES</i> genes in a bicistronic <i>groESL</i> operon. Among 4,861 completely-sequenced prokaryotic genomes, 884 possess duplicate <i>groEL</i> genes and 770 possess <i>groEL</i> genes with no neighboring <i>groES</i>. It is unclear whether stand-alone <i>groEL</i> requires <i>groES</i> in order to function and, if required, how duplicate <i>groEL</i> genes and unequal <i>groES</i> genes balance their expressions. In <i>Myxococcus xanthus</i> DK1622, we determined that, while duplicate <i>groEL</i>s were alternatively deletable, the single <i>groES</i> that clusters with <i>groEL1</i> was essential for cell survival. Either GroEL1 or GroEL2 required interactions with GroES for <i>in vitro</i> and <i>in vivo</i> functions. Deletion of <i>groEL1</i> or <i>groEL2</i> resulted in decreased expressions of both <i>groEL</i> and <i>groES</i>; and ectopic complementation of <i>groEL</i> recovered not only the <i>groEL</i> but also <i>groES</i> expressions. The addition of an extra <i>groES</i> gene upstream <i>groEL2</i> to form a bicistronic operon had almost no influence on <i>groES</i> expression and the cell survival rate, whereas over-expression of <i>groES</i> using a self-replicating plasmid simultaneously increased the <i>groEL</i> expressions. The results indicated that <i>M. xanthus</i> DK1622 cells coordinate expressions of the duplicate <i>groEL</i> and single <i>groES</i> genes for synergistic functions of GroELs and GroES. We proposed a potential regulation mechanism for the expression coordination.
Project description:Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step in the Calvin-Benson cycle, which transforms atmospheric carbon into a biologically useful carbon source. The slow catalytic rate of Rubisco and low substrate specificity necessitate the production of high levels of this enzyme. In order to engineer a more efficient plant Rubisco, we need to better understand its folding and assembly process. Form I Rubisco, found in green algae and vascular plants, is a hexadecamer composed of 8 large subunits (RbcL), encoded by the chloroplast genome and 8 small, nuclear-encoded subunits (RbcS). Unlike its cyanobacterial homolog, which can be reconstituted in vitro or in E. coli, assisted by bacterial chaperonins (GroEL-GroES) and the RbcX chaperone, biogenesis of functional chloroplast Rubisco requires Cpn60-Cpn20, the chloroplast homologs of GroEL-GroES, and additional auxiliary factors, including Rubisco accumulation factor 1 (Raf1), Rubisco accumulation factor 2 (Raf2) and Bundle sheath defective 2 (Bsd2). The discovery and characterization of these factors paved the way for Arabidopsis Rubisco assembly in E. coli. In the present review, we discuss the uniqueness of hetero-oligomeric chaperonin complex for RbcL folding, as well as the sequential or concurrent actions of the post-chaperonin chaperones in holoenzyme assembly. The exact stages at which each assembly factor functions are yet to be determined. Expression of Arabidopsis Rubisco in E. coli provided some insight regarding the potential roles for Raf1 and RbcX in facilitating RbcL oligomerization, for Bsd2 in stabilizing the oligomeric core prior to holoenzyme assembly, and for Raf2 in interacting with both RbcL and RbcS. In the long term, functional characterization of each known factor along with the potential discovery and characterization of additional factors will set the stage for designing more efficient plants, with a greater biomass, for use in biofuels and sustenance.