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Tadashi Maruyama - One of the best experts on this subject based on the ideXlab platform.
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Natural chaperonin of the hyperthermophilic archaeum, Thermococcus strain KS-1: a hetero-oligomeric chaperonin with variable subunit composition
Molecular Microbiology, 2004Co-Authors: Takao Yoshida, Masafumi Yohda, Akira Ideno, Shuichi Hiyamuta, Tadashi MaruyamaAbstract:To study the difference in expression of the chaperonin α- and β-subunits in Thermococcus strain KS-1 (T. KS-1), we measured their intracellular contents at various growth temperatures using subunit-specific antibodies. The β-subunit was significantly more abundant with increasing temperature (maximum at 93°C), whereas the α-subunit was not. Native PAGE with Western blot analysis indicated that the natural Chaperonins in the crude extracts of T. KS-1 cells grown between 65°C and 95°C migrate as single bands with different mobility. The recombinant α- and β-subunit homo-oligomers migrated differently from each other and from natural Chaperonins. Immunoprecipitation also showed that the natural chaperonin was the hetero-oligomer. These results indicate that chaperonin in T. KS-1 formed a hetero-oligomer with variable subunit composition, and that the β-subunit may be adapted to a higher temperature than the α-subunit. T. KS-1 probably changes its chaperonin subunit composition to acclimatize to the ambient temperature.
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archaeal group ii chaperonin mediates protein folding in the cis cavity without a detachable groes like co chaperonin
Journal of Molecular Biology, 2002Co-Authors: Takao Yoshida, Hideki Taguchi, Masafumi Yohda, Tadashi Maruyama, Rika Kawaguchi, Masasuke Yoshida, Takuo Yasunaga, Takeyuki WakabayashiAbstract: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 α or β-subunit chaperonin homo-oligomer (α16mer and β16mer) from a hyperthermoplilic archaeum, Thermococcus strain KS-1, using a model substrate, green fluorescent protein (GFP). The α16mer and β16mer 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 β16mer but not by the α16mer. A mutant α-subunit chaperonin homo-oligomer (trap-α) could capture the non-native protein but lacked the ability to refold it. Although trap-α suppressed ATP-dependent refolding of GFP mediated by α16mer or β16mer, it did not affect the AMP-PNP-dependent refolding. This indicated that the GFP refolding mediated by β16mer with AMP-PNP was not accessible to the trap-α. Gel filtration chromatography and a protease protection experiment revealed that this refolded GFP, in the presence of AMP-PNP, was associated with β16mer. After the completion of GFP refolding mediated by β16mer with AMP-PNP, addition of ATP induced an additional refolding of GFP. Furthermore, the β16mer 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.
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Chaperonin in a Thermophilic Methanogen, Methanococcus Thermolithotrophicus
New Developments in Marine Biotechnology, 1998Co-Authors: Masahiro Furutani, Toshii Iida, Shigeyuki Yamano, Tadashi MaruyamaAbstract:Molecular chaperones play an important role in the protein foldings in vivo. Chaperonin is a 60 kDa major member of molecular chaperones and has two types, Group I and Group II. The Group I chaperonin is GroEL-like complex in eubacteria, mitochondria and chloroplasts. Whereas archaeral chaperonin which is coiled thermosomc and eukaryotic cytosol TCP- I protein belong to Group II (Trent et al., 1991). While a chaperonin was purified from a hyperthermophilic methanogen, Methanopyrus kandleri (Andra et al., 1996), biochemical and functional characters of Chaperonins in methanogens have remained to be clarified. We report here characteristics of chaperonin of a thermophilic methanogen, Methanococcus thermolithotrophicus.
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Chaperonin in a Thermophilic Methanogen, Methanococcus Thermolithotrophicus
New Developments in Marine Biotechnology, 1998Co-Authors: Masahiro Furutani, Toshii Iida, Shigeyuki Yamano, Tadashi MaruyamaAbstract:Molecular chaperones play an important role in the protein foldings in vivo. Chaperonin is a 60 kDa major member of molecular chaperones and has two types, Group I and Group II. The Group I chaperonin is GroEL-like complex in eubacteria, mitochondria and chloroplasts. Whereas archaeral chaperonin which is coiled thermosomc and eukaryotic cytosol TCP- I protein belong to Group II (Trent et al., 1991). While a chaperonin was purified from a hyperthermophilic methanogen, Methanopyrus kandleri (Andra et al., 1996), biochemical and functional characters of Chaperonins in methanogens have remained to be clarified. We report here characteristics of chaperonin of a thermophilic methanogen, Methanococcus thermolithotrophicus.
Ulrich F Hartl - One of the best experts on this subject based on the ideXlab platform.
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differential substrate specificity of group i and group ii Chaperonins in the archaeon methanosarcina mazei
Molecular Microbiology, 2009Co-Authors: Angela Hirtreiter, Giulia Calloni, Francesca Forner, Burghardt Scheibe, Magda Puype, Joel Vandekerckhove, Matthias Mann, Ulrich F HartlAbstract:: Chaperonins are macromolecular machines that assist in protein folding. The archaeon Methanosarcina mazei has acquired numerous bacterial genes by horizontal gene transfer. As a result, both the bacterial group I chaperonin, GroEL, and the archaeal group II chaperonin, thermosome, coexist. A proteome-wide analysis of chaperonin interactors was performed to determine the differential substrate specificity of GroEL and thermosome. At least 13% of soluble M. mazei proteins interact with Chaperonins, with the two systems having partially overlapping substrate sets. Remarkably, chaperonin selectivity is independent of phylogenetic origin and is determined by distinct structural and biochemical features of proteins. GroEL prefers well-conserved proteins with complex alpha/beta domains. In contrast, thermosome substrates comprise a group of faster-evolving proteins and contain a much wider range of different domain folds, including small all-alpha and all-beta modules, and a greater number of large multidomain proteins. Thus, the group II Chaperonins may have facilitated the evolution of the highly complex proteomes characteristic of eukaryotic cells.
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coexistence of group i and group ii Chaperonins in the archaeon methanosarcina mazei
Journal of Biological Chemistry, 2003Co-Authors: Daniel Klunker, Uwe Deppenmeier, Angela Hirtreiter, Luis Figueiredo, Dean J Naylor, Gunter Pfeifer, Volker Muller, Bernd Haas, Gerhard Gottschalk, Ulrich F HartlAbstract:Abstract Two distantly related classes of cylindrical chaperonin complexes assist in the folding of newly synthesized and stress-denatured proteins in an ATP-dependent manner. Group I Chaperonins are thought to be restricted to the cytosol of bacteria and to mitochondria and chloroplasts, whereas the group II Chaperonins are found in the archaeal and eukaryotic cytosol. Here we show that members of the archaeal genus Methanosarcina co-express both the complete group I (GroEL/GroES) and group II (thermosome/prefoldin) chaperonin systems in their cytosol. These mesophilic archaea have acquired between 20 and 35% of their genes by lateral gene transfer from bacteria. In Methanosarcina mazei Go1, both Chaperonins are similarly abundant and are moderately induced under heat stress. The M. mazei GroEL/GroES proteins have the structural features of their bacterial counterparts. The thermosome contains three paralogous subunits, α, β, and γ, which assemble preferentially at a molar ratio of 2:1:1. As shown in vitro, the assembly reaction is dependent on ATP/Mg2+ or ADP/Mg2+ and the regulatory role of the β subunit. The co-existence of both chaperonin systems in the same cellular compartment suggests the Methanosarcina species as useful model systems in studying the differential substrate specificity of the group I and II Chaperonins and in elucidating how newly synthesized proteins are sorted from the ribosome to the proper chaperonin for folding.
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coexistence of group i and group ii Chaperonins in the archaeon methanosarcina mazei
Journal of Biological Chemistry, 2003Co-Authors: Daniel Klunker, Uwe Deppenmeier, Angela Hirtreiter, Luis Figueiredo, Dean J Naylor, Gunter Pfeifer, Volker Muller, Bernd Haas, Gerhard Gottschalk, Ulrich F HartlAbstract:Two distantly related classes of cylindrical chaperonin complexes assist in the folding of newly synthesized and stress-denatured proteins in an ATP-dependent manner. Group I Chaperonins are thought to be restricted to the cytosol of bacteria and to mitochondria and chloroplasts, whereas the group II Chaperonins are found in the archaeal and eukaryotic cytosol. Here we show that members of the archaeal genus Methanosarcina co-express both the complete group I (GroEL/GroES) and group II (thermosome/prefoldin) chaperonin systems in their cytosol. These mesophilic archaea have acquired between 20 and 35% of their genes by lateral gene transfer from bacteria. In Methanosarcina mazei Go1, both Chaperonins are similarly abundant and are moderately induced under heat stress. The M. mazei GroEL/GroES proteins have the structural features of their bacterial counterparts. The thermosome contains three paralogous subunits, alpha, beta, and gamma, which assemble preferentially at a molar ratio of 2:1:1. As shown in vitro, the assembly reaction is dependent on ATP/Mg2+ or ADP/Mg2+ and the regulatory role of the beta subunit. The co-existence of both chaperonin systems in the same cellular compartment suggests the Methanosarcina species as useful model systems in studying the differential substrate specificity of the group I and II Chaperonins and in elucidating how newly synthesized proteins are sorted from the ribosome to the proper chaperonin for folding.
Masafumi Yohda - One of the best experts on this subject based on the ideXlab platform.
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Bridging human chaperonopathies and microbial Chaperonins.
Communications Biology, 2019Co-Authors: Everly Conway De Macario, Masafumi Yohda, Alberto J L Macario, Frank T. RobbAbstract:Chaperonins are molecular chaperones that play critical physiological roles, but they can be pathogenic. Malfunctional Chaperonins cause chaperonopathies of great interest within various medical specialties. Although the clinical-genetic aspects of many chaperonopathies are known, the molecular mechanisms causing chaperonin failure and tissue lesions are poorly understood. Progress is necessary to improve treatment, and experimental models that mimic the human situation provide a promising solution. We present two models: one prokaryotic (the archaeon Pyrococcus furiosus) with eukaryotic-like Chaperonins and one eukaryotic (Chaetomium thermophilum), both convenient for isolation-study of Chaperonins, and report illustrative results pertaining to a pathogenic mutation of CCT5. Everly Conway de Macario et al. present a review of microbial models of human chaperonopathies. They discuss the recent progress in using microbes to model human pathogenic mutations and to elucidate disease mechanisms.
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eLS - Thermosome: A Group II Chaperonin of Archaea
eLS, 2016Co-Authors: Yohei Y. Yamamoto, Masafumi YohdaAbstract:Chaperonin, heat shock protein 60, plays an important role in the proteostasis of cytosol. Chaperonins are divided into two groups: group I and group II. Group II Chaperonins exist in eukaryotes and archaea, and these Chaperonins are named CCT/TRiC and Thermosome, respectively. Group II Chaperonins have almost the same structure as group I Chaperonins. The main difference is the existence of a helical protrusion, which constitutes a built-in lid of the cavity. Group II chaperonin captures an unfolded protein in the cavity in the open conformation and changes to the closed conformation in an ATP-dependent manner, which triggers folding of the captured protein. As Thermosome is relatively stable and simple compared with CCT, the conformational change mechanism and also the interaction with co-chaperone, Prefoldin, have been studied in detail using Thermosome. The conformational change procedure of Thermosome will give insights on protein folding mechanism by group II Chaperonins. Key Concepts Group II chaperonin is an essential cytosolic molecular chaperone in eukaryotes and archaea. The archaeal group II chaperonin is named Thermosome. Group II chaperonin has a built-in lid for the central cavity. As ATP induces a conformational change from the open to the closed conformation, twisting of the ring occurs. Group II chaperonin cooperates with a co-chaperone, Prefoldin, which captures an unfolded protein and transfers it to the central cavity of the group II chaperonin. Keywords: Hsp60; chaperone; chaperonin; Thermosome; conformational change; protein folding; ATPase
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inter ring communication is dispensable in the reaction cycle of group ii Chaperonins
Journal of Molecular Biology, 2014Co-Authors: Yohei Y. Yamamoto, Noriyuki Ishii, Yuki Abe, Kazuki Moriya, Mayuno Arita, Keiichi Noguchi, Hiroshi Sekiguchi, Yuji C Sasaki, Masafumi YohdaAbstract:Abstract Chaperonins are ubiquitous molecular chaperones with the subunit molecular mass of 60 kDa. They exist as double-ring oligomers with central cavities. An ATP-dependent conformational change of the cavity induces the folding of an unfolded protein that is captured in the cavity. In the group I Chaperonins, which are present in eubacteria and eukaryotic organelles, inter-ring communication takes important role for the reaction cycle. However, there has been limited study on the inter-ring communication in the group II Chaperonins that exist in archaea and the eukaryotic cytosol. In this study, we have constructed the asymmetric ring complex of a group II chaperonin using circular permutated covalent mutants. Although one ring of the asymmetric ring complex lacks ATPase or ATP binding activity, the other wild-type ring undergoes an ATP-dependent conformational change and maintains protein-folding activity. The results clearly demonstrate that inter-ring communication is dispensable in the reaction cycle of group II Chaperonins.
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functional characterization of recombinant prefoldin complexes from a hyperthermophilic archaeon thermococcus sp strain ks 1
Journal of Molecular Biology, 2008Co-Authors: Ryo Iizuka, Yuri Sugano, Tadayuki Imanaka, Takao Yoshida, Akashi Ohtaki, Shinsuke Fujiwara, Masafumi YohdaAbstract: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.
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Characterization of archaeal group II chaperonin-ADP-metal fluoride complexes: Implications that group II Chaperonins operate as a "two-stroke engine"
Journal of Biological Chemistry, 2005Co-Authors: Ryo Iizuka, Takao Yoshida, Noriyuki Ishii, Tamotsu Zako, Kazunobu Takahashi, Kosuke Maki, Tomonao Inobe, Kunihiro Kuwajima, Masafumi YohdaAbstract:Group II Chaperonins, found in Archaea and in the eukaryotic cytosol, act independently of a cofactor corresponding to GroES of group I Chaperonins. Instead, the helical protrusion at the tip of the apical domain forms a built-in lid of the central cavity. Although many studies on the lid's conformation have been carried out, the conformation in each step of the ATPase cycle remains obscure. To clarify this issue, we examined the effects of ADP-aluminum fluoride (AlFx) and ADP-beryllium fluoride (BeFx) complexes on alpha-chaperonin from the hyperthermophilic archaeum, Thermococcus sp. strain KS-1. Biochemical assays, electron microscopic observations, and small angle x-ray scattering measurements demonstrate that alpha-chaperonin incubated with ADP and BeFx exists in an asymmetric conformation; one ring is open, and the other is closed. The result indicates that alpha-chaperonin also shares the inherent functional asymmetry of bacterial and eukaryotic cytosolic Chaperonins. Most interestingly, addition of ADP and BeFx induced alpha-chaperonin to encapsulate unfolded proteins in the closed ring but did not trigger their folding. Moreover, alpha-chaperonin incubated with ATP and AlFx or BeFx adopted a symmetric closed conformation, and its functional turnover was inhibited. These forms are supposed to be intermediates during the reaction cycle of group II Chaperonins.
A D Miller - One of the best experts on this subject based on the ideXlab platform.
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refolding and recognition of mitochondrial malate dehydrogenase by escherichia coli Chaperonins cpn 60 groel and cpn10 groes
Biochemical Journal, 1994Co-Authors: J P Hutchinson, T S Elthaher, A D MillerAbstract: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.
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escherichia coli Chaperonins cpn60 groel and cpn10 groes do not catalyse the refolding of mitochondrial malate dehydrogenase
Biochemical Journal, 1993Co-Authors: A D Miller, Karim Maghlaoui, G Albanese, D A Kleinjan, C SmithAbstract: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.
Peter A Lund - One of the best experts on this subject based on the ideXlab platform.
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Chaperonin Abundance Boosts Bacterial Fitness
2020Co-Authors: C. M. Santosh Kumar, Shekhar C. Mande, Kritika Chugh, Anirban Dutta, Vishnuvardhan Mahamkali, Tungadri Bose, Sharmila S. Mande, Peter A LundAbstract:The ability of Chaperonins to buffer mutations that affect the protein folding pathways of suggests that their abundance should be evolutionarily advantageous. Here, we investigate the effect of chaperonin overproduction on cellular fitness in Escherichia coli. We demonstrate that chaperonin abundance confers (a) an ability to tolerate higher temperatures, (b) improved cellular fitness and (c) enhanced folding of metabolic enzymes, which is expected to lead to enhanced energy harvesting potential.
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replacement of groel in escherichia coli by the group ii chaperonin from the archaeon methanococcus maripaludis
Journal of Bacteriology, 2016Co-Authors: Riddhi Shah, Andrew T Large, Astrid Ursinus, Preethy Gowrinathan, Jorg Martin, Peter A LundAbstract:ABSTRACT Chaperonins are required for correct folding of many proteins. They exist in two phylogenetic groups: group I, found in bacteria and eukaryotic organelles, and group II, found in archaea and eukaryotic cytoplasm. The two groups, while homologous, differ significantly in structure and mechanism. The evolution of group II Chaperonins has been proposed to have been crucial in enabling the expansion of the proteome required for eukaryotic evolution. In an archaeal species that expresses both groups of Chaperonins, client selection is determined by structural and biochemical properties rather than phylogenetic origin. It is thus predicted that group II Chaperonins will be poor at replacing group I Chaperonins. We have tested this hypothesis and report here that the group II chaperonin from Methanococcus maripaludis (Mm-cpn) can partially functionally replace GroEL, the group I chaperonin of Escherichia coli. Furthermore, we identify and characterize two single point mutations in Mm-cpn that have an enhanced ability to replace GroEL function, including one that allows E. coli growth after deletion of the groEL gene. The biochemical properties of the wild-type and mutant Mm-cpn proteins are reported. These data show that the two groups are not as functionally diverse as has been thought and provide a novel platform for genetic dissection of group II Chaperonins. IMPORTANCE The two phylogenetic groups of the essential and ubiquitous Chaperonins diverged approximately 3.7 billion years ago. They have similar structures, with two rings of multiple subunits, and their major role is to assist protein folding. However, they differ with regard to the details of their structure, their cofactor requirements, and their reaction cycles. Despite this, we show here that a group II chaperonin from a methanogenic archaeon can partially substitute for the essential group I chaperonin GroEL in E. coli and that we can easily isolate mutant forms of this chaperonin with further improved functionality. This is the first demonstration that these two groups, despite the long time since they diverged, still overlap significantly in their functional properties.
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Identification of Elements That Dictate the Specificity of Mitochondrial Hsp60 for Its Co-Chaperonin
2016Co-Authors: Avital Parnas, Peter A Lund, Shahar Nisemblat, Celeste Weiss, Galit Levy-rimler, Amir Pri-or, Tsaffrir Zor, Peter Bross, Abdussalam AzemAbstract: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
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Identification of elements that dictate the specificity of mitochondrial Hsp60 for its co-chaperonin.
PLoS ONE, 2012Co-Authors: Avital Parnas, Peter A Lund, Shahar Nisemblat, Celeste Weiss, Galit Levy-rimler, Amir Pri-or, Tsaffrir Zor, Peter Bross, Abdussalam AzemAbstract: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.
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multiple Chaperonins in bacteria why so many
Fems Microbiology Reviews, 2009Co-Authors: Peter A LundAbstract:A significant proportion of bacteria express two or more chaperonin genes. Chaperonins are a group of molecular chaperones, defined by sequence similarity, required for the folding of some cellular proteins. Chaperonin monomers have a mass of c. 60 kDa, and are typically found as large protein complexes containing 14 subunits arranged in two rings. The mechanism of action of the Escherichia coli GroEL protein has been studied in great detail. It acts by binding to unfolded proteins and enabling them to fold in a protected environment where they do not interact with any other proteins. GroEL can assist the folding of many proteins of different sizes, sequences, and structures, and homologues from many different bacteria can functionally replace GroEL in E. coli. What then are the functions of multiple Chaperonins? Do they provide a mechanism for cells to increase their general chaperoning ability, or have they become specialized to take on specific novel cellular roles? Here I will review the genetic, biochemical, and phylogenetic evidence that has a bearing on this question, and show that there is good evidence for at least some specificity of function in multiple chaperonin genes.