Two kinds of archaeal group II chaperonin subunits with different thermostability in Thermococcus strain KS-1

The thermostability of the recombinant alpha- and beta-subunit homo-oligomers (alpha16mer and beta16mer) and of natural chaperonins purified from cultured Thermococcus strain KS-1 cells was measured to understand the mechanism for the thermal acclimatization of T. KS-1. The beta-subunit content of the natural chaperonin from cells grown at 90 degrees C was higher than that at 80 degrees C. The optimum temperature for ATPase activity of the natural chaperonins was 80-90 degrees C, whereas that for alpha16mer and beta16mer was 60 degrees C and over 90 degrees C respectively. Judging from the ATPase activity, beta16mer was more thermostable than alpha16mer. The thermostabilities of the natural chaperonins were intermediate between alpha16mer and beta16mer, whereas the natural chaperonin with a higher beta-subunit content was more stable than that with a lower beta-subunit content. Native polyacrylamide gel electrophoresis (PAGE) revealed that the chaperonin oligomers thermally dissociated to their ATPase-inactive monomers. The thermal denaturation process monitored by circular dichroism showed that the free beta-subunit was more stable than the free alpha-subunit, and that the secondary structure of the chaperonin monomer in the oligomer was more stable than that in the free monomer. These results suggest that the structure of these subunits was stabilized in the oligomer, and that an increase in the beta-subunit content conferred higher thermostability to the natural hetero-oligomeric chaperonin.     

Nucleotide specificity of an archaeal group II chaperonin from Thermococcus strain KS-1 with reference to the ATP-dependent protein folding cycle

The archaeal chaperonin-mediated folding of green fluorescent protein (GFP) was examined in the presence of various nucleotides. The recombinant alpha- and beta-subunit homo-oligomers and natural chaperonin oligomer from Thermococcus strain KS-1 exhibited folding activity with not only ATP but also with CTP, GTP, or UTP. The ADP-bound form of both recombinant and natural chaperonin had the ability to capture non-native GFP, but could not refold it in the presence of CTP, GTP or UTP until ATP was supplied. The archaeal chaperonin thus utilized ATP, but could not use other nucleoside triphosphates in the cytoplasm where ADP was present.     

Archaeal group II chaperonin mediates protein folding in the cis-cavity without a detachable GroES-like co-chaperonin.

Group II chaperonins of archaea and eukaryotes are distinct from group I chaperonins of bacteria. Whereas group I chaperonins require the co-chaperonin Cpn-10 or GroES for protein folding, no co-chaperonin has been known for group II. The protein folding mechanism of group II chaperonins is not yet clear. To understand this mechanism, we examined protein refolding by the recombinant alpha or beta-subunit chaperonin homo-oligomer (alpha16mer and beta16mer) from a hyperthermoplilic archaeum, Thermococcus strain KS-1, using a model substrate, green fluorescent protein (GFP). The alpha16mer and beta16mer captured the non-native GFP and promoted its refolding without any co-chaperonin in an ATP dependent manner. A non-hydrolyzable ATP analog, AMP-PNP, induced the GFP refolding mediated by beta16mer but not by the alpha16mer. A mutant alpha-subunit chaperonin homo-oligomer (trap-alpha) could capture the non-native protein but lacked the ability to refold it. Although trap-alpha suppressed ATP-dependent refolding of GFP mediated by alpha16mer or beta16mer, it did not affect the AMP-PNP-dependent refolding. This indicated that the GFP refolding mediated by beta16mer with AMP-PNP was not accessible to the trap-alpha. Gel filtration chromatography and a protease protection experiment revealed that this refolded GFP, in the presence of AMP-PNP, was associated with beta16mer. After the completion of GFP refolding mediated by beta16mer with AMP-PNP, addition of ATP induced an additional refolding of GFP. Furthermore, the beta16mer preincubated with AMP-PNP showed the ability to capture the non-native GFP. These suggest that AMP-PNP induced one of two chaperonin rings (cis-ring) to close and induced protein refolding in this ring, and that the other ring (trans-ring) could capture the unfolded GFP which was refolded by adding ATP. The present data indicate that, in the group II chaperonin of Thermococcus strain KS-1, the protein folding proceeds in its cis-ring in an ATP-dependent fashion without any co-chaperonin.     

Construction and characterization of the hetero-oligomer of the group II chaperonin from the hyperthermophilic archaeon, Thermococcus sp. strain KS-1

The hyperthermophilic archaeon Thermococcus sp. strain KS-1 (T. KS-1) expresses two different chaperonin subunits, α and β, for the folding of its proteins. The composition of the subunits in the hexadecameric double ring changes with temperature. The content of the β subunit significantly increases according to the increase in temperature. The homo-oligomer of the β subunit, Cpnβ, is more thermostable than that of the α subunit, Cpnα. Since Cpnα and Cpnβ also have different protein folding activities and interactions with prefoldin, the hetero-oligomer is thought to exhibit different characteristics according to the content of subunits. The hetero-oligomer of the T. KS-1 chaperonin has not been studied, however, because the α and β subunits form hetero-oligomers of varying compositions when they are expressed simultaneously. In this study, we characterized the T. KS-1 chaperonin hetero-oligomer, Cpnαβ, containing both α and β in the alternate order, which was constructed by the expression of α and β subunits in a coordinated fashion and protease digestion. Cpnαβ protected citrate synthase from thermal aggregation, promoted the folding of acid-denatured GFP in an ATP-dependent manner, and exhibited an ATP-dependent conformational change. The yield of refolded GFP generated by Cpnαβ was almost equivalent to that generated by Cpnβ but lower than that generated by Cpnα. In contrast, Cpnαβ exhibited almost the same level of thermal stability as Cpnα, which was lower than that of Cpnβ. The affinity of Cpnαβ to prefoldin was found to be between those of Cpnα and Cpnβ, as expected.     

Functional characterization of the recombinant group II chaperonin alpha from Thermoplasma acidophilum

The functional characteristics of group II chaperonins, especially those from archaea, have not been elucidated extensively. Here, we performed a detailed functional characterization of recombinant chaperonin alpha subunits (16-mer) (Ta-cpn alpha) from the thermophilic archaea Thermoplasma acidophilum as a model protein of archaeal group II chaperonins. Recombinant Ta-cpn alpha formed an oligomeric ring structure similar to that of native protein, and displayed an ATP hydrolysis activity (optimal temperature: 60 degrees C) in the presence of either magnesium, manganese or cobalt ions. Ta-cpn alpha was able to bind refolding intermediates of Thermus MDH and GFP in the absence of ATP, and to promote the refolding of Thermus MDH at 50 degrees C in the presence of Mg2+-, Mn2+-, or Co2+-ATP. Ta-cpn alpha also prevented thermal aggregation of rhodanese and luciferase at 50 degrees C. Interestingly, Ta-cpn alpha in the presence of Mn2+ ion showed an increased hydrophobicity, which correlated with an increased efficiency in substrate protein binding. Our finding that Ta-cpn alpha chaperonin system displays folding assistance ability with ATP-dependent substrate release may provide a detailed look at the potential functional capabilities of archaeal chaperonins.     

An engineered chaperonin caging a guest protein: Structural insights and potential as a protein expression tool

The structure of a chaperonin caging a substrate protein is not quite clear. We made engineered group II chaperonins fused with a guest protein and analyzed their structural and functional features. Thermococcus sp. KS-1 chaperonin alpha-subunit (TCP) which forms an eightfold symmetric double-ring structure was used. Expression plasmids were constructed which carried two or four TCP genes ligated head to tail in phase and a target protein gene at the 3' end of the linked TCP genes. Electron microscopy showed that the expressed gene products with the molecular sizes of ~120 kDa (di-TCP) and ~230 kDa (tetra-TCP) formed double-ring complexes similar to those of wild-type TCP. The tetra-TCP retained ATPase activity and its thermostability was significantly higher than that of the wild type. A 260-kDa fusion protein of tetra-TCP and green fluorescent protein (GFP, 27 kDa) was able to form the double-ring complexes with green fluorescence. Image analyses indicated that the GFP moiety of tetra-TCP/GFP fusion protein was accommodated in the central cavity, and tetra-TCP/GFP formed the closed-form similar to that crystallographically resolved in group II chaperonins. Furthermore, it was suggested that caging GFP expanded the cavity around the bottom. Using this tetra-TCP fusion strategy, two virus structural proteins (21-25 kDa) toxic to host cells or two antibody fragments (25-36 kDa) prone to aggregate were well expressed in the soluble fraction of Escherichia coli. These fusion products also assembled to double-ring complexes, suggesting encapsulation of the guest proteins. The antibody fragments liberated by site-specific protease digestion exhibited ligand-binding activities.     

The lower hydrolysis of ATP by the stress protein GroEL is a major factor responsible for the diminished chaperonin activity at low temperature

The chaperonins GroEL and GroES were shown to facilitate the refolding of urea-unfolded rhodanese in an ATP-dependent process at 25 or 37 degrees C. A diminished chaperonin activity was observed at 10 degrees C, however. At low temperature, GroEL retains its ability to form a complex with urea-unfolded rhodanese or with GroES. GroEL is also able to bind ATP at 10 degrees C. Interestingly, the ATPase activity of GroEL was highly decreased at low temperatures. Hydrolysis of ATP by GroEL was 60% less at 10 degrees C than at 25 degrees C. We conclude that the reduced hydrolysis of ATP by GroEL is a major but perhaps not the only factor responsible for the diminished chaperonin activity at 10 degrees C. GroEL may function primarily at higher temperatures in which the ability of GroEL to hydrolyze ATP is not compromised.     

Cooperativity of alpha- and beta-subunits of group II chaperonin from the hyperthermophilic archaeum Aeropyrum pernix K1

alpha- and beta-subunits (ApCpnA and ApCpnB) are group II chaperonins from the hyperthermophilic archaeum Aeropyrum pernix K1, specialized in preventing the aggregation and inactivation of substrate proteins under conditions of transient heat stress. In the present study, the cooperativity of alpha- and beta-subunits from the A. pernix K1 was investigated. The ApCpnA and ApCpnB chaperonin genes were overexpressed in E. coli Rosetta and Codonplus (DE3), respectively. Each of the recombinant alpha- and beta- subunits was purified to 92% and 94% by using anionexchange chromatography. The cooperative activity between purified alpha- and beta-subunits was examined using citrate synthase (CS), alcohol dehydrogenase (ADH), and malate dehydrogenase (MDH) as substrate proteins. The addition of both alpha- and beta-subunits could effectively protect CS and ADH from thermal aggregation and inactivation at 43 degreesC and 50 degreesC, respectively, and MDH from thermal inactivation at 80 degreesC and 85 degreesC. Moreover, in the presence of ATP, the protective effects of alpha- and beta-subunits on CS from thermal aggregation and inactivation, and ADH from thermal aggregation, were more enhanced, whereas cooperation between chaperonins and ATP in protection activity on ADH and MDH (at 85 degreesC) from thermal inactivation was not observed. Specifically, the presence of both alpha- and beta- subunits could effectively protect MDH from thermal inactivation at 80 degreesC in an ATP-dependent manner.     

Crystal structures of the group II chaperonin from Thermococcus strain KS-1: steric hindrance by the substituted amino acid, and inter-subunit rearrangement between two crystal forms

The crystal structures of the group II chaperonins consisting of the alpha subunit with amino acid substitutions of G65C and/or I125T from the hyperthermophilic archaeum Thermococcus strain KS-1 were determined. These mutants have been shown to be active in ATP hydrolysis but inactive in protein folding. The structures were shown to be double-ring hexadecamers in an extremely closed form, which was consistent with the crystal structure of native alpha8beta8-chaperonin from Thermoplasma acidophilum. Comparisons of the present structures with the atomic structures of the GroEL14-GroES7-(ADP)7 complex revealed that the deficiency in protein-folding activity with the G65C amino acid substitution is caused by the steric hindrance of the local conformational change in an equatorial domain. We concluded that this mutant chaperonin with G65C substitution is deprived of the smooth conformational change in the refolding-reaction cycle. We obtained a new form of crystal with a distinct space group at a lower concentration of sulfate ion in the presence of nucleotide. The crystal structure obtained at the lower concentration of sulfate ion tilts outward, and has much looser inter-subunit contacts compared with those in the presence of a higher concentration of sulfate ion. Such subunit rotation has never been characterized in group II chaperonins. The crystal structure obtained at the lower concentration of sulfate ion tilts outward, and has much looser inter-subunit contacts compared with those in the presence of a higher concentration of sulfate ion.     

Genetic, enzymatic, and structural analyses of phenylalanyl-tRNA synthetase from Thermococcus kodakaraensis KOD1

Phenylalanyl-tRNA synthetase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 (Tk-PheRS) was cloned. The open reading frames for both the alpha-subunit (Tk-pheRSA) and beta-subunit (Tk-pheRSB) genes were 1,503 bp (501 amino acids) and 1,722 bp (574 amino acids), respectively. Tk-pheRSB located 879 bp downstream from Tk-pheRSA with a putative TATA box, suggesting that these two subunits are transcribed and regulated independently in KOD1 cells. Tk-PheRS and its respective subunits were expressed in Escherichia coli cells and the proteins were purified. Tk-PheRS showed an optimum enzymatic activity at around 95 degrees C and retained its tertiary structure at 98 degrees C. The estimated isoelectric point (pI) for the alpha-subunit is 9.4 and that for the beta-subunit is 4.6, the largest difference among the 12 kinds of PheRSs reported. The considerable thermostability of Tk-PheRS may be responsible for the electrostatic interaction between the alpha- and beta-subunits.     

ATP binding is critical for the conformational change from an open to closed state in archaeal group II chaperonin

Group II chaperonins, found in archaea and in eukaryotic cytosol, do not have a co-chaperonin corresponding to GroES. Instead, it is suggested that the helical protrusion extending from the apical domain acts as a built-in lid for the central cavity and that the opening and closing of the lid is regulated by ATP binding and hydrolysis. However, details of this conformational change remain unclear. To investigate the conformational change associated with the ATP-driven cycle, we conducted protease sensitivity analyses and tryptophan fluorescence spectroscopy of alpha-chaperonin from a hyperthermophilic archaeum, Thermococcus strain KS-1. In the nucleotide-free or ADP-bound state, the chaperonin, especially in the helical protrusion region, was highly sensitive to proteases. Addition of ATP and ammonium sulfate induced the transition to the relatively protease-resistant form. The fluorescence intensity of the tryptophan residue introduced at the tip of the helical protrusion was enhanced by the presence of ATP or ammonium sulfate. We conclude that ATP binding induces the conformational change from the lid-open to lid-closed form in archaeal group II chaperonin.     

ATPase cycle of an archaeal chaperonin

Recent structural data imply differences in allosteric behavior of the group I chaperonins, typified by GroEL from Escherichia coli, and the group II chaperonins, which comprise archaeal thermosome and eukaryotic TRiC/CCT. Therefore, this study addresses the mechanism of interaction of adenine nucleotides with recombinant alpha-only and native alphabeta-thermosomes from Thermoplasma acidophilum, which also enables us to analyze the role of the heterooligomeric composition of the natural thermosome. Although all subunits of the alpha-only thermosome seem to bind nucleotides tightly and independently, the native chaperonin has two different classes of ATP-binding sites. Furthermore, for the alpha-only thermosome, the steady-state ATPase rate is determined by the cleavage reaction itself, whereas, for the alphabeta-thermosome, the rate-limiting step is associated with a post-hydrolysis isomerisation into a non-covalent ADP*P(i) species prior to the release of the gamma-phosphate group. After half-saturation with ATP, a negative cooperativity in hydrolysis is observed for both thermosomes. The effect of Mg(2+) and K(+) nucleotide cycling is documented. We conclude that archaeal chaperonins have unique allosteric properties and discuss them in the light of the mechanism established for the group I chaperonins.     

Chaperonins facilitate the in vitro folding of monomeric mitochondrial rhodanese.

In vitro refolding of the monomeric mitochondrial enzyme, rhodanese (thiosulfate sulfurtransferase; EC 2.8.1.1) is facilitated by molecular chaperonins. The four components: two proteins from Escherichia coli, chaperonin 60 (groEL) and chaperonin 10 (groES), MgATP, and K+, are necessary for the in vitro folding of rhodanese. These were previously shown to be necessary for the in vitro folding of ribulose-1,5-bisphosphate carboxylase at temperatures in excess of 25 degrees C (Viitanen, P. V., Lubben, T. H., Reed, J., Goloubinoff, P., O'Keefe, D. P., and Lorimer, G. H. (1990) Biochemistry 29, 5665-5671). The labile folding intermediate, rhodanese-I, which rapidly aggregates at 37 degrees C in the absence of the chaperonins, can be stabilized by forming a binary complex with chaperonin 60. The discharge of the binary chaperonin 60-rhodanese-I complex, results in the formation of active rhodanese, and requires the presence of chaperonin 10. Optimal refolding is associated with a K(+)-dependent hydrolysis of ATP. At lower protein concentrations and 25 degrees C, where aggregation is reduced, a fraction of the rhodanese refolds to an active form in the absence of the chaperonins. This spontaneous refolding can be arrested by chaperonin 60. There is some refolding (approximately equal to 20%) when ATP is replaced by nonhydrolyzable analogs, but there is no refolding in the presence of ADP or AMP. ATP analogs may interfere with the interaction of rhodanese-I with the chaperonins. Nondenaturing detergents facilitate rhodanese refolding by interacting with exposed hydrophobic surfaces of folding intermediates and thereby prevent aggregation (Tandon, S., and Horowitz, P. (1986) J. Biol. Chem. 261, 15615-15618). The chaperonin proteins appear to play a similar role in as much as they can replace the detergents. Consistent with this view, chaperonin 60, but not chaperonin 10, binds 2-3 molecules of the hydrophobic fluorescent reporter, 1,1'-bi(4-anilino)naphthalene-S,5'-disulfonic acid, indicating the presence of hydrophobic surfaces on chaperonin 60. The number of bound probe molecules is reduced to 1-2 molecules when chaperonin 10 and MgATP are added. The results support a model in which chaperonins facilitate folding, at least in part, by interacting with partly folded intermediates, thus preventing the interactions of hydrophobic surfaces that lead to aggregation.     

Functional characterization of recombinant prefoldin complexes from a hyperthermophilic archaeon, Thermococcus sp. strain KS-1

Prefoldin is a heterohexameric molecular chaperone complex that is found in the eukaryotic cytosol and also in archaea. It captures a nonnative protein and subsequently delivers it to a group II chaperonin for proper folding. Archaeal prefoldin is a heterocomplex containing two α subunits and four β subunits with the structure of a double β-barrel assembly, with six long coiled coils protruding from it like a jellyfish with six tentacles. We have studied the protein folding mechanism of group II chaperonin using those of Thermococcus sp. strain KS-1 (T. KS-1) because they exhibit high protein folding activity in vitro. We have also demonstrated functional cooperation between T. KS-1 chaperonins and prefoldin from Pyrococcus horikoshii OT3. Recent genome analysis has shown that Thermococcus kodakaraensis KOD1 contains two pairs of prefoldin subunit genes, correlating with the existence of two different chaperonin subunits. In this study, we characterized four different recombinant prefoldin complexes composed of two pairs of prefoldin subunits (α1, α2, β1, and β2) from T. KS-1. All of them (α1-β1, α2-β1, α1-β2, and α2-β2) exist as α₂β₄ heterohexamers and can protect several proteins from forming aggregates with different activities. We have also compared the collaborative activity between the prefoldin complexes and the cognate chaperonins. Prefoldin complexes containing the β1 subunit interacted with the chaperonins more strongly than those with the β2 subunit. The results suggest that Thermococcus spp. express different prefoldins for different substrates or conditions as chaperonins.     

Purification and characterization of chaperonin 10 from Chromatium vinosum.

Chromatium vinosum contains a polypeptide that is functionally and structurally similar to the Escherichia coli chaperonin 10. The protein has been purified to homogeneity by sucrose density gradient centrifugation followed by gel filtration using a Bio-Gel A-1.5 m column. The molecular mass of chaperonin 10, as determined by gel filtration or nondenaturing polyacrylamide gel electrophoresis, is 95 kDa. The oligomer is composed of seven or eight subunits. Comparisons of the overall amino acid composition and N-terminal sequences among chaperonin 10 species from C. vinosum and E. coli reflect a high degree of similarity. A physical association between chaperonins 60 and 10 from C. vinosum, in vitro, is supported by three experimental approaches. First, the proteins form a stable binary complex in sucrose density gradients, gel filtration chromatography, and nondenaturing polyacrylamide gel electrophoresis, solely in the presence of ATP and Mg2+. Second, chaperonin 10 from C. vinosum binds, selectively, to a chaperonin 60-coupled Affi-Gel 10 matrix column. Third, a slight molar excess of chaperonin 10 is able to abolish, almost completely, the ATPase in chaperonin 60. The rate for ATPase activity of chaperonin 60 from C. vinosum is enhanced when supplemented with monovalent cations.     

Single-molecule fluorescence polarization study of conformational change in archaeal group II chaperonin

Group II chaperonins found in archaea and in eukaryotic cytosol mediate protein folding without a GroES-like cofactor. The function of the cofactor is substituted by the helical protrusion at the tip of the apical domain, which forms a built-in lid on the central cavity. Although many studies on the change in lid conformation coupled to the binding and hydrolysis of nucleotides have been conducted, the molecular mechanism of lid closure remains poorly understood. Here, we performed a single-molecule polarization modulation to probe the rotation of the helical protrusion of a chaperonin from a hyperthermophilic archaeum, Thermococcus sp. strain KS-1. We detected approximately 35° rotation of the helical protrusion immediately after photorelease of ATP. The result suggests that the conformational change from the open lid to the closed lid state is responsible for the approximately 35° rotation of the helical protrusion.     

Non-functioning chaperonin GroEL stimulates protein aggregation

To clarify the role of chaperones in the development of amyloid diseases, the interaction of the chaperonin GroEL with misfolded proteins and recombinant prions has been studied. The efficiency of the chaperonin-assisted folding of denatured glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was shown to be decreased in the presence of prions. Prions are capable of binding to GroEL immobilized on Sepharose, but this does not prevent the interaction between GroEL and other denatured proteins. The size of individual proteins (GroEL, GAPDH, and the recombinant prion) and aggregates formed after their mixing have been determined by the dynamic light scattering analysis. It was shown that at 25°C, the non-functioning chaperonin (equimolar mixture of GroEL and GroES in the absence of Mg-ATP) bound prion yielding large aggregates (greater than 400 nm). The addition of Mg-ATP decreased significantly the size of the aggregates to 70–80 nm. After blocking of one of the chaperonin active sites by oxidized denatured GAPDH, the aggregate size increased to 1200 nm, and the addition of Mg-ATP did not prevent the aggregation. These data indicate the significant role of chaperonins in the formation of amyloid structures and demonstrate the acceleration of aggregation in the presence of functionally inactive chaperonins. The suggested model can be used for the analysis of the efficiency of antiaggregants in the system containing chaperonins.     

The plastid chaperonin

The discovery and properties of the plastid chaperonin are described. This chaperonin is implicated in the folding and assembly of the enzyme ribulose bisphosphate carboxylase-oxygenase and in the folding of proteins imported into plastids from the cytosol. The plastid chaperonin appears to be unique in that it contains two distinct types of 60 kDa polypeptide, whereas only a single subunit type has been reported for the bacterial and mitochondrial chaperonins. The plastid chaperonin polypeptides are encoded by nuclear genes which are subject to complex regulation.     

Glycine at the 65th position plays an essential role in ATP-dependent protein folding by Archael group II chaperonin.

In the previous study, we have found that G65C and I125T double mutant of alpha chaperonin homo-oligomer from a hyperthermophilic archaeum, Thermococcus sp. strain KS-1, lacks ATP-dependent protein refolding activity despite showing ATPase activity and the ability to bind the denatured proteins. In this study, we have characterized several mutant Thermococcus chaperonin homo-oligomers with the amino acid substitutions of Gly-65 or Ile-125. The results showed that amino acid residue at 65th position should be a small amino acid such as glycine or alanine for the ATP-dependent refolding activity. The alpha chaperonin homo-oligomers with amino acid substitution of Gly-65 by amino acids whose side chains are larger than the methyl group did not have ATP-dependent protein refolding activity, but exhibited an increase of the binding affinity for unfolded proteins in the presence of ATP or AMP-PNP. (c)2001 Elsevier Science.     

Mammalian mitochondrial chaperonin 60 functions as a single toroidal ring.

Chaperonins are thought to participate in the process of protein folding in bacteria and in eukaryotic mitochondria and chloroplasts. While some chaperonins are relatively well characterized, the structures of the mammalian chaperonins are unknown. We have expressed a mammalian mitochondrial chaperonin 60 in Escherichia coli and purified the recombinant protein to homogeneity. Structural and biochemical analyses of this protein establish a single toroidal structure of seven subunits, in contrast to the homologous bacterial, fungal, and plant chaperonin 60s, which have double toroidal structures comprising two layers of seven identical subjects each. The recombinant mammalian chaperonin 60, together with the mammalian chaperonin 10 (but not with bacterial chaperonin 10), facilitates the formation of catalytically active ribulose-bisphosphate carboxylase from an unfolded state in the presence of K+ and MgATP. Analysis of the partial reactions involved in this in vitro reconstitution reveals that the single toroid of chaperonin 60 can form stable complexes with both unfolded or partially folded [35S]ribulose-bisphosphate carboxylase and mitochondrial (but not bacterial) chaperonin 10 in the presence of MgATP. We conclude that the minimal functional unit of chaperonin 60 is a single hepatmeric toroid.