Transient kinetic analysis of ATP-induced allosteric transitions in the eukaryotic chaperonin containing TCP-1

The chaperonin CCT (chaperonin containing t-complex polypeptide 1 (TCP-1)) from bovine testis was mixed rapidly with different concentrations of ATP and the time-resolved change in fluorescence emission, upon excitation at 280 nm, was followed. Two kinetic phases were observed and assigned by (i) analyzing the dependence of the corresponding observed rate constants on ATP concentration; and (ii) by carrying out mixing experiments also with ADP, ATPgammaS and ATP without K(+). The values of the observed rate constants corresponding to both phases are found to be dependent on ATP concentration. The observed rate constant corresponding to the fast phase displays a bi-sigmoidal dependence on ATP concentration with Hill coefficients that are similar to those determined in steady-state ATPase experiments. This phase most likely reflects ATP binding-induced conformational changes. The rate constant of the conformational change in the presence of excess ATP is about 17s(-1) (at 25 degrees C) and is tenfold slower than the corresponding rate constant of GroEL. The observed rate constant corresponding to the second slower phase displays a hyperbolic dependence on ATP concentration. This phase is not observed in mixing experiments of CCT with ADP, ATPgammaS or ATP without K(+) and it, therefore, reflects a conformational change associated with ATP hydrolysis. Taken together, our results indicate that the kinetic mechanism of the allosteric transitions of CCT differs considerably from that of GroEL.     

Nested allosteric interactions in the cytoplasmic chaperonin containing TCP-1

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.     

Characterization of the cytoplasmic chaperonin containing TCP-1 from the Antarctic fish Notothenia coriiceps

The cytoplasmic chaperonin containing TCP-1 (CCT) plays a critically important role in the folding and biogenesis of many cytoskeletal proteins, including tubulin and actin. For marine ectotherms, the chronically cold Southern Ocean (−2 to +2°C) poses energetic challenges to protein folding, both at the level of substrate proteins and with respect to the chaperonin/chaperone folding system. Here we report the partial functional and structural characterization of CCT from an Antarctic notothenioid fish, Notothenia coriiceps. We find that the mechanism of folding by the Antarctic fish CCT differed from that of mammalian CCT: (1) the former complex was able to bind denatured β-tubulin but (2) when reconstituted with rabbit Cofactor A, failed to release the protein to yield the tubulin/cofactor intermediate. Moreover, the amino acid sequences of the N. coriiceps CCT β and θ chains contained residue substitutions in the equatorial, apical, and intermediate domains that would be expected to increase the flexibility of the subunits, thus facilitating function of the chaperonin in an energy poor environment. Our work contributes to the growing realization that protein function in cold-adapted organisms reflects a delicate balance between the necessity of structural flexibility for catalytic activity and the concomitant hazard of cold-induced denaturation.     

Review: nucleotide-dependent conformational changes of the chaperonin containing TCP-1

Current biochemical and structural studies on the conformational changes induced by the nature of nucleotide bound to the chaperonin containing testis complex polypeptide 1 (CCT) are examined to see how consistent the data are. This exercise suggests that the biochemical and structural data are in good agreement. CCT clearly appears as a folding nano-machine fueled by ATP. A careful comparison of the biochemical and structural data, however, highlights a number of points that remain to be carefully documented in order to better understand the nature of the conformational changes in CCT that yield folded target proteins. Special effort should be made to clearly answer the points listed at the end of this review in order to obtain the dynamic sequence of events yielding folded proteins in the eukaryotic cytoplasm similar to what has been obtained for prokaryotes.     

Reversible interaction of beta-actin along the channel of the TCP-1 cytoplasmic chaperonin.

The cytoplasm of eukaryotes contains a heteromeric toroidal chaperonin assembled from the t-complex TCP-1 and several other related polypeptides. The structure of the TCP-1 cytoplasmic chaperonin and that of the binary complex formed between this chaperonin and unfolded beta-actin have been studied using electron microscopy and image processing techniques. Two-dimensional averaging of front views reveals a circular stain-excluding mass surrounding a central stain-penetrating region in which the stain is excluded upon actin binding. Sections of a three-dimensional reconstruction of the chaperonin show that the inner core is an empty channel that becomes filled upon binary complex formation with unfolded beta-actin. Upon incubation with Mg-ATP, the beta-actin:chaperonin complex discharges the actin such that the chaperonin central cavity reappears. Side views from different forms of TCP-1 reveals that upon Mg-ATP binding, the cytoplasmic chaperonin undergoes a structural rearrangement that is confirmed using a new classification method.     

Activities of the chaperonin containing TCP-1 (CCT): implications for cell cycle progression and cytoskeletal organisation.

The chaperonin containing TCP-1 (CCT) is required for the production of native actin and tubulin and numerous other proteins, several of which are involved in cell cycle progression. The mechanistic details of how CCT acts upon its folding substrates are intriguing: whilst actin and tubulin bind in a sequence-specific manner, it is possible that some proteins could use CCT as a more general binding interface. Therefore, how CCT accommodates the folding requirements of its substrates, some of which are produced in a cell cycle-specific manner, is of great interest. The reliance of folding substrates upon CCT for the adoption of their native structures results in CCT activity having far-reaching implications for a vast array of cellular processes. For example, the dependency of the major cytoskeletal proteins actin and tubulin upon CCT results in CCT activity being linked to any cellular process that depends on the integrity of the microfilament and microtubule-based cytoskeletal systems.     

Chaperonin-containing TCP-1 complex directly binds to the cytoplasmic domain of the LOX-1 receptor

Lectin-like oxidized low-density lipoprotein receptor (LOX-1) is a scavenger receptor that binds oxidized low-density lipoprotein (OxLDL) and has a role in atherosclerosis development. The N-terminus intracellular region (cytoplasmic domain) of LOX-1 mediates receptor internalization and trafficking, potentially through intracellular protein interactions. Using affinity isolation, we identified 6 of the 8 components of the chaperonin-containing TCP-1 (CCT) complex bound to LOX-1 cytoplasmic domain, which we verified by coimmunoprecipitation and immunostaining in human umbilical vein endothelial cells. We found that the interaction between CCT and LOX-1 is direct and ATP-dependent and that OxLDL suppressed this interaction. Understanding the association between LOX-1 and the CCT complex may facilitate the design of novel therapies for cardiovascular disease.     

Developmental and light-dependent changes of the cytosolic chaperonin containing TCP-1 (CCT) subunits in maize seedlings, and the localization in coleoptiles

The cytosolic chaperonin containing TCP-1 (CCT) is known to keep fold cytoskeletal proteins and is involved in the proper organization of the cytoskeleton. These studies are based on the assumption that growth responses linked to structural rearrangement of the plant cytoskeleton include the action of CCT and the need for newly synthesized tubulin. The presence of the α- and ɛ- subunits of CCT was investigated in soluble fractions of protein extracts from maize mesocotyls and coleoptiles at distinct growth stages. The CCT-subunits, tubulins and actin decreased in the coleoptile in response to far-red light. In addition, independent from light treatment, the amount of CCTɛ abundance declined with age in coleoptiles and mesocotyls between 2 and 4.5 days after sowing. In contrast to CCTɛ, no significant light regulation of CCTα was found in the mesocotyl. In two day old, light-grown rapidly elongating coleoptiles part of the CCTα subunit and the bulk of actin and tubulin was found shifted into fractions of high molecular weight complexes when compared to slowly elongating, dark grown coleoptiles. In 4.5 day old, etiolated and elongating coleoptiles, part of both CCT-subunits and cytoskeleton proteins were found in fractions of high molecular weight. A complete disappearance of these polypeptides was observed in old far-red irradiated growth-arrested coleoptiles. CCTɛ was found to be co-localized to microtubular structures and to the nucleus. We conclude from our data that abundance of CCT-subunits in soluble extracts is dependent on age and light treatment, but independent from the growth stage of mesocotyl and coleoptile.     

The t complex polypeptide 1 (TCP-1) is associated with the cytoplasmic aspect of Golgi membranes

The t complex polypeptide 1 (TCP-1) is a protein of unknown function expressed in large amounts during spermatogenesis. Rat monoclonal antibodies recognizing TCP-1 have been prepared and used to immunoprecipitate and Western blot a 57 kd protein from germ cell and tissue culture cell extracts. In tissue culture cells, indirect immunofluorescent localization of antigen indicated a perinuclear distribution similar to that of the Golgi apparatus. Analysis of the TCP-1 distribution in tissue culture cells showed that the polypeptide was associated with the cytoplasmic aspect of membranes of the trans-Golgi network (TGN). The distribution in spermatids suggested that TCP-1 was localized to structures often associated with the developing acrosome. The TCP-1 antigenic epitopes are highly conserved, allowing the protein to be identified in cells across a wide variety of vertebrate species and tissues. These experiments suggest that TCP-1 may be essential for transport of proteins through the exocytic pathway in all cells and required in large amounts for acrosome formation in developing spermatids.     

Hetero‐oligomeric CPN60 resembles highly symmetric group‐I chaperonin structure revealed by Cryo‐EM

The chloroplast chaperonin system is indispensable for the biogenesis of Rubisco, the key enzyme in photosynthesis. Using Chlamydomonas reinhardtii as a model system, we found that in vivo the chloroplast chaperonin consists of CPN60α, CPN60β1 and CPN60β2 and the co‐chaperonin of the three subunits CPN20, CPN11 and CPN23. In Escherichia coli, CPN20 homo‐oligomers and all possible other chloroplast co‐chaperonin hetero‐oligomers are functional, but only that consisting of CPN11/20/23‐CPN60αβ1β2 can fully replace GroES/GroEL under stringent stress conditions. Endogenous CPN60 was purified and its stoichiometry was determined to be 6:2:6 for CPN60α:CPN60β1:CPN60β2. The cryo‐EM structures of endogenous CPN60αβ1β2/ADP and CPN60αβ1β2/co‐chaperonin/ADP were solved at resolutions of 4.06 and 3.82 Å, respectively. In both hetero‐oligomeric complexes the chaperonin subunits within each ring are highly symmetric. Through hetero‐oligomerization, the chloroplast co‐chaperonin CPN11/20/23 forms seven GroES‐like domains, which symmetrically interact with CPN60αβ1β2. Our structure also reveals an uneven distribution of roof‐forming domains in the dome‐shaped CPN11/20/23 co‐chaperonin and potentially diversified surface properties in the folding cavity of the CPN60αβ1β2 chaperonin that might enable the chloroplast chaperonin system to assist in the folding of specific substrates.     

Novel isolation method and structural stability of a eukaryotic chaperonin: the TCP-1 ring complex from rabbit reticulocytes.

In the course of removing a contaminant from preparations of aminoacyl-tRNA synthetase complexes, a novel purification method has been developed for the eukaryotic cytoplasmic chaperonin known as TRiC or CCT. This method uses only three steps: ammonium sulfate precipitation, pelleting into a sucrose cushion, and heparin-agarose chromatography. As judged by electrophoresis, sedimentation, and electron microscopy, the preparations are homogeneous. The particle is identified as a chaperonin from electrophoretic polypeptide pattern, electron microscopic images, direct mass measurement by sedimentation velocity analysis, amino-terminal sequencing, and ATP-dependent refolding of rhodanese and actin. Further investigation of the biochemical and physical properties of the particle demonstrates that its constituent polypeptides are not glycosylated. The particle as a whole binds strongly to polyanionic matrices. Of particular note is that negatively stained images of chaperonin adsorbed to a single carbon layer are distinctly different from those where it is sandwiched between two layers. In the former, the "characteristic" ring and four-stripe barrel predominate. In the latter, most images are round with a highly reticulated surface, the average particle diameter increases from 15 to 18 nm, and additional side, end, and substrate-containing views are observed. The particle structure is strikingly resistant to physical forces (long-term storage, repeated cycles of freezing and thawing, sedimentation), detergents (Triton, deoxycholate), salts (molar levels of KCl or LiCl), and pH changes (9-6). Only a strongly chaotropic salt (NaSCN) and extremely acidic conditions (pH 4.5) cause aggregation and dissociation of TRiC, respectively. However, treatment with KCl or deoxycholate reduces TRiC folding activity.     

Multiple equilibria of the Escherichia coli chaperonin GroES revealed by mass spectrometry

Nanospray time-of-flight mass spectrometry has been used to study the assembly of the heptamer of the Escherichia coli cochaperonin protein GroES, a system previously described as a monomer-heptamer equilibrium. In addition to the monomers and heptamers, we have found measurable amounts of dimers and hexamers, the presence of which suggests the following mechanism for heptamer assembly: 2 Monomers <--> Dimer; 3 Dimers <--> Hexamer; Hexamer + Monomer <--> Heptamer. Equilibrium constants for each of these steps, and an overall constant for the Monomer <--> Heptamer equilibrium, have been estimated from the data. These constants imply a standard free-energy change, DeltaG(0), of about 9 kcal/mol for each contact surface formed between GroES subunits, except for the addition of the last subunit, where DeltaG(0) = 6 kcal/mol. This lower value probably reflects the loss of entropy when the heptamer ring is formed. These experiments illustrate the advantages of electrospray mass spectrometry as a method of measuring all components of a multiple equilibrium system.     

ATP-induced structural change in myosin subfragment-1 revealed by the location of protease cleavage sites on the primary structure

To understand the nature of the ATP-induced structural change in myosin subfragment-1, rabbit and chicken skeletal subfragments-1s were cleaved by various proteolytic enzymes in the absence, and in the presence, of ATP and the exact locations of the cleavage sites that were affected by ATP were determined from the amino end analysis of fragments by the use of a protein sequencer. It was found that subtilisin cleaved a site between Gln27 and Asn28 of rabbit subfragment-1 and between Gln28 and Asn29 of chicken subfragment-1 only in the presence of ATP. Thermolysin cleaved a site between Pro31 and Phe32 of chicken subfragment-1 in the presence of ATP, but the same site of rabbit subfragment-1 was not cleaved. The location of these sites is quite similar to the ATP-induced chymotryptic cleavage site of chicken gizzard heavy meromyosin, between Trp29 and Ser30 as reported by others. It is suggested, therefore, that the structure and the ATP-induced structural change in the regions are similar in these subfragment-1s. ATP also changes the cleavage rate of the 26K-50K junction by many proteases. Exact cleavage sites were determined and the relationship between their location and the suppression or the enhancement by ATP of the cleavage was studied. It was found that the cleavage sites were restricted to a quite narrow region and only the cleavage by thermolysin that attacked the middle of the region was enhanced by ATP. The distribution of the cleavage sites and the effect of ATP suggest that ATP induces drastic structural change at the middle of the 26K-50K junction region. The region attacked easily by many proteases coincided very well with a hydrophilic region indicated by the hydropathy index. The region probably protrudes outside and is, therefore, easily attacked by many proteases.     

Evolution of the chaperonin families (HSP60, HSP 10 and TCP-1) of proteins and the origin of eukaryotic cells

The members of the 10 kDa and 60 kDa heat-shock chaperonin proteins (Hsp10 and Hsp60 or Cpn10 and Cpn60), which form an operon in bacteria, are present in all eubacteria and eukaryotic ceil organelles such as mitochondria and chloroplasts. In archaebacteria and eukaryotic cell cytosol, no close homologues of Hsp10 or Hsp60 have been identified. However, these species (or ceil compartments) contain the Tcp-1 family of proteins (distant homologues of Hsp60). Phylogenetic analysis based on global alignments of Hsp60 and Hsp10 sequences presented here provide some evidence regarding the evolution of mitochondria from a member of the α-subdivision of Gram-negative bacteria and chloroplasts from cyanobacterial species, respectively. This inference is strengthened by the presence of sequence signatures that are uniquely shared between Hsp60 homologues from α-purple bacteria and mitochondria on one hand, and the chloroplasts and cyanobacterial hsp60s on the other. Within the α-purple subdivision, species such as Rickettsia and Ehrlichia, which live intracellularly within eukaryotic cells, are indicated to be the closest relatives of mitochondrial Homologues, In the Hsp60 evolutionary tree, rooted using the Tcp-1 homologue, the order of branching of the major groups was as follows: Gram-positive bacteria — cyanobacteria and chloroplasts — chlamydiae and spirochaetes —β and γ-Gram-negative purple bacteria —α-purple bacteria — mitochondria. A similar branching order was observed independently in the Hsp10 tree. Multiple Hsp60 homologues, when present in a group of species, were found to be clustered together in the trees, indicating that they evolved by independent gene-duplication events. This review also considers in detail the evolutionary relationship between Hsp50 and Tcp-1 families of proteins based on two different models (viz. archaebacterial and chimeric) for the origin of eukaryotic cell nucleus. Some predictions of the chimeric model are also discussed.     

A novel cochaperonin that modulates the ATPase activity of cytoplasmic chaperonin

The folding of alpha- and beta-tubulin requires three proteins: the heteromeric TCP-1-containing cytoplasmic chaperonin and two additional protein cofactors (A and B). We show that these cofactors participate in the folding process and do not merely trigger release, since in the presence of Mg-ATP alone, alpha- and beta-tubulin target proteins are discharged from cytoplasmic chaperonin in a nonnative form. Like the prokaryotic cochaperonin GroES, which interacts with the prototypical Escherichia coli chaperonin GroEL and regulates its ATPase activity, cofactor A modulates the ATPase activity of its cognate chaperonin. However, the sequence of cofactor A derived from a cloned cDNA defines a 13-kD polypeptide with no significant homology to other known proteins. Moreover, while GroES functions as a heptameric ring, cofactor A behaves as a dimer. Thus, cofactor A is a novel cochaperonin that is structurally unrelated to GroES.     

A kinetic analysis of the nucleotide-induced allosteric transitions in a single-ring mutant of GroEL

The function of GroE requires a complex system of allosteric communication driven by protein-nucleotide interactions. These rearrangements couple the binding and hydrolysis of ATP to an overall reaction cycle in which substrate proteins are bound, encapsulated and released. Positive cooperativity with respect to ATP binding occurs within one heptameric ring of GroEL, while negative cooperativity between the two rings generates an inherent asymmetry between the two rings. A previously engineered mutant of GroEL in which the ring-ring contacts are broken gives rise to a single-ring version of the wild-type chaperonin (SR1). We have studied the kinetics of the nucleotide-induced conformational changes in a single-tryptophan variant of SR1 (Y485W-SR1) and compared the resulting data with those we reported previously for the double-ring, single-tryptophan variant of wild-type GroEL (Y485W-GroEL). Remarkably, the parting of the rings does not have a major effect on the conformational changes occurring within the heptameric ring and a kinetic model is presented to describe the sequence of structural rearrangements that occur upon ATP binding to the SR1 molecule. The observation that both the ATP-induced and ADP-induced conformational rearrangements occur more rapidly in the SR1 than they do in wild-type GroEL, indicates that intra-ring conformational changes in the double-ring structure must overcome conformational constraints provided by the presence of the second ring. Lastly, the data presented here imply a role for inter-ring allostery in controlling the dissociation-association behaviour of the GroES co-protein in the overall reaction cycle.     

Factors controlling fibroblast growth factor receptor-1's cytoplasmic trafficking and its regulation as revealed by FRAP analysis

Biochemical and microscopic studies have indicated that FGFR1 is a transmembrane and soluble protein present in the cytosol and nucleus. How FGFR1 enters the cytosol and subsequently the nucleus to control cell development and associated gene activities has become a compelling question. Analyses of protein synthesis, cytoplasmic subcompartmental distribution and movement of FGFR1-EGFP and FGFR1 mutants showed that FGFR1 exists as three separate populations (a) a newly synthesized, highly mobile, nonglycosylated, cytosolic receptor that is depleted by brefeldin A and resides outside the ER-Golgi lumen, (b) a slowly diffusing membrane receptor population, and (c) an immobile membrane pool increased by brefeldin A. RSK1 increases the highly mobile cytosolic FGFR1 population and its overall diffusion rate leading to increased FGFR1 nuclear accumulation, which coaccumulates with RSK1. A model is proposed in which newly synthesized FGFR1 can enter the (a) "nuclear pathway," where the nonglycosylated receptor is extruded from the pre-Golgi producing highly mobile cytosolic receptor molecules that rapidly accumulate in the nucleus or (b) "membrane pathway," in which FGFR1 is processed through the Golgi, where its movement is spatially restricted to trans-Golgi membranes with limited lateral mobility. Entrance into the nuclear pathway is favored by FGFR1's interaction with kinase active RSK1.