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J. Biol. Chem., Vol. 279, Issue 30, 31788-31795, July 23, 2004
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From the
Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16, Naka-cho, Koganei, Tokyo 184-8588, Japan, the ¶Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo Bunkyo-ku, Tokyo 113-8656, Japan, the ||Department of Physics, School of Science and Engineering, Waseda University, 3-4-1 Okubo, Tokyo 169-8555, Japan, and the **Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada
Received for publication, March 15, 2004 , and in revised form, May 6, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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sub-unit are important for molecular chaperone activity and for the interaction with a chaperonin. Our data are consistent with substrate and chaperonin binding sites on prefoldin that are different but in close proximity, which suggests a possible handover mechanism of prefoldin substrates to the chaperonin. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The group II chaperonins cooperate with a co-chaperone, prefoldin/GimC (19–21). Prefoldin (PFD) has been shown to participate in the maturation of actin and members of the tubulin family by transferring them in the incompletely folded states to CCT. Although there is neither actin nor tubulin in archaea, both prefoldin homologues and group II chaperonins have invariably been identified in all archaeal species (21, 22). Archaeal prefoldin consists of only two species of subunits, namely 
and , whereas eukaryotic prefoldins are composed of two different but related class subunits and four related class subunits. The crystal structure of the archaeal prefoldin from Methanobacterium thermoautotrophicum (MtPFD) has been determined at a resolution of 2.3 Å (23). It resembles a jellyfish in that its body consists of a double -barrel assembly with six long tentacle-like coiled-coils protruding from it. The distal regions of the coiled-coils partially expose hydrophobic patches, and there is evidence that coiled-coils are required for the multivalent binding of non-native proteins (23). Eukaryotic prefoldin possesses a structure similar to that of archaeal prefoldin and has been observed to capture a substrate protein (actin) in the cavity formed by its six "tentacles" (24). In the three-dimensional reconstitution of the prefoldin·actin complex, actin appears to interact with prefoldin in a region that is not well defined but is also consistent with the distal regions of the coiled-coils (24). Additional evidence for actin and tubulin substrate interaction with the distal ends of different but overlapping sets of eukaryotic prefoldin subunits has been obtained recently (25).
In contrast, the interaction and functional cooperation between the archaeal chaperonin and prefoldin is comparably less understood. The archaeal chaperones represent a powerful model system for examining their functional cooperation because of their simple subunit composition and structural stability.
We have already reported that the prefoldin from Pyrococcus horikoshii OT3 (PhPFD) functionally cooperates with chaperonins in the refolding of the green fluorescent protein (GFP) in vitro (26). In the present study, we have examined the interaction between a prefoldin and a chaperonin from hyperthermophilic archaea by immunoprecipitation, single molecule observation, and surface plasmon resonance, and characterized the substrate and chaperonin binding site of prefoldin. Our results reveal that both the distal N- and C-terminal regions of the subunit are important for substrate binding and also for interaction with a chaperonin. Importantly, the dual function of the prefoldin tips (substrate and chaperonin binding) may represent an important property that facilitates the functional cooperation with a chaperonin.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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for the preparation of plasmids and BL21(DE3) for expression. Site-directed mutagenesis was performed using a QuikChange site-directed mutagenesis kit (Stratagene). Restriction enzymes, Ex TaqDNA polymerase and other reagents for gene manipulation were purchased from Takara Bio Inc. (Shiga, Japan). Citrate synthase (CS) from porcine heart, bovine serum albumin, glucose oxidase, and catalase were purchased from Sigma. All other chemicals were of analytical grade, and solutions were made up in ultrapure water.
Proteins—Both wild-type and mutated PhPFDs were prepared by reconstitution from purified subunits. Truncation mutant genes were prepared by PCR amplification from the wild-type gene using the primers (wt, PH0527Fw 5'-CAT-ATG-ATA-AGG-ATG-GCT-CAG-AA-3', PH0527Rv 5'-GGA-TCC-CTAC-TTC-TTA-ACC-TTA-AAG-C-3'; tc8, PH0527Fw, PH0527tc8Rv 5'-TTG-GAT-CCC-TAA-CTT-TGC-TTT-TGC-TGT-A-3'; tn17, PH0527tn17Fw 5'CAT-ATG-TAC-CAG-GTT-TTA-CAA-GCT-CAA-G-3', PH0527Rv; wt, PH0532Fw 5'-CAT-ATG-CAG-AAC-ATT-CCT-CCC-CA-3', PH0532Rv 5'-GTC-GAC-TCA-GCC-AGC-GGT-AGG-CGG-CC-3'; tc5, PH0532Fw, PH0532tc5Rv 5'-GTC-GAC-TCA-CCT-CAG-AGC-GGC-TTG-AAT-CTT-3'; tc6, PH0532Fw, PH0532tc6Rv 5'-GTC-GAC-TCA-CAG-AGC-GGC-TTG-AAT-CTT-CTG-3'; tc7, PH0532Fw, PH0532tc7Rv 5'-GTC-GAC-TCA-AGC-GGC-TTG-AAT-CTT-CTG-AG-3'; tc8, PH0532Fw, PH0532tc8Rv 5'-TTG-TCG-ACT-CAG-GCT-TGA-ATC-TTC-TGA-G-3'; tn10, PH0532tn10Fw 5'-CAT-ATG-CTT-GGC-CAA-CTC-GAT-ACG-TAT-C-3', PH0532Rv). The constructs were designated according to the terminus, number of residues, and subunit type that was mutagenized, as exemplified by tc8, a mutant subunit with an 8-amino acid truncation from the C terminus, and tn17, a mutant subunit of with a 17-amino acid truncation from the N terminus. The amplified DNA for the truncation mutant was cloned into pT7Blue T. After sequence confirmation, the gene was excised with NdeI and BamHI ( subunit) or NdeI and SalI ( subunit), and inserted into pET23b. A mutant subunit, L111A, denoting an amino acid substitution of Leu111 by Ala, was prepared by a QuikChange site-directed mutagenesis kit using the primers (PH0532L111AFw 5'-GAT-TCA-AGC-CGC-TGC-GAG-GCC-GCC-TAC-CGC-3', PH0532L111ARv 5'-GCG-GTA-GGC-GGC-CTC-GCA-GCG-GCT-TGA-ATC-3'). The construct was also verified by DNA sequencing.
Each subunit was expressed in E. coli BL21(DE3). After removal of most E. coli proteins by heat treatment at 80 °C for 30 min, it was purified by anion exchange chromatography on a DEAE-Toyopearl column (Tosoh, Tokyo, Japan) using buffer A (50 mM Tris-HCl, pH 8.0, 0.1 mM EDTA) with a gradient of NaCl, and subsequent gel filtration on a HiLoad 26/60 Superdex 200 prep grade column (Amersham Biosciences) equilibrated with buffer A containing 150 mM NaCl. PhPFD complexes were reconstituted by incubating the mixture of the purified and subunits at the molar ratio of 1:2 at 80 °C for 30 min. The reconstituted complex was purified by gel filtration on a Bio-Prep column (Bio-Rad). The purified complex contained and subunits at the molar ratio of 1:2 as judged by SDS-PAGE analysis.
Chaperonins from Pyrococcus horikoshii OT3 (PhCPN), Thermococcus sp strain KS-1 (TkCPN), and Thermus thermophilus HB8 (TthCPN) were expressed and purified as described previously (17, 26).2 Wild-type prefoldin from M. thermoautotrophicum, MtPFD, was prepared as described previously (22).
The GFP used in this report is a heat-stable mutant, with alanine inserted in the N-terminal region, a His tag in the C-terminal region, and amino acid substitution of F99S, M153T, V163A, and L165F (5, 27). It was purified as described previously (28).
Immunoprecipitation of Prefoldin with Anti-chaperonin Antibody— The immunoprecipitation experiments were performed using IMMU-NOcatcher (CytoSignal) to examine the interactions between the prefoldin and the chaperonin in the presence or absence of denatured GFP. The anti-TkCPN antibody was used for the immunoprecipitation. Because of the high sequence homology between TkCPN and PhCPN, the anti-TkCPN antibody can specifically immunoprecipitate PhCPN. The preparation and purification of the antibody were described previously (29). The sample solutions for the immunoprecipitation containing 7.0 µM PhPFD and 10 mM HCl-unfolded GFP (at the specified concentration) were mixed and incubated at 65 °C for 10 min prior to the addition of 1.4 µM PhCPN. The samples were incubated at 65 °C for an additional 10 min. Then, 10 µl of anti-TkCPN antibody was added to 210 µl of the sample solution and incubated for 1 h at room temperature. Protein A/G resin (10 µl) was then applied to the mixture and incubated for 45 min. Proteins bound to the resin were collected by centrifugation, washed, and resuspended in SDS-PAGE sample solution. After incubation for 15 min, the bound proteins were collected by centrifugation and then analyzed by SDS-PAGE.
Preparation of Fluorescent-labeled Proteins—We prepared a mutant TkCPN with amino acid replacement of Asn472 by cysteine (TkCPN472C). This mutation site was selected on the basis of the criterion that the residue is fully exposed to solvent, minimizing the effect on the function of the chaperonin. TkCPN472C was treated with 5 mM dithiothreitol to reduce the cysteine residue. Dithiothreitol was removed by gel filtration on a Sephadex G-25 column (Amersham Biosciences) equilibrated with buffer B (25 mM HEPES-KOH, pH 7.4, 100 mM KCl, 5 mM MgCl2). TkCPN472C was labeled with Alexa Fluor 488 C5 maleimide (Alexa488, Molecular Probes) for 30 min at room temperature. PhPFD was labeled with Cy5 Mono-reactive Dye (Amersham Biosciences) by incubation for 1 h at room temperature. Amino groups of PhPFD were modified with Cy5. The labeled TkCPN472C (488-TkCPN) and PhPFD (Cy5-PhPFD) were separated from the unreacted reagents by gel filtration. The extent of labeling was determined by adsorption spectroscopy. The molar ratios of Alexa488 to TkCPN (16-mer) and that of Cy5 to PhPFD (6-mer) were 2.6 and 0.9, respectively. The 488 labeled TkCPN showed the same ATPase activity as that of the non-labeled TkCPN472C (data not shown). Cy5-PhPFD exhibited nearly the same activity in protecting CS from thermally induced aggregation compared with non-labeled PFD (data not shown).
Single-molecule Imaging of CPN·PFD Complex—100 nM 488-TkCPN was incubated with 100 nM Cy5-PhPFD at 60 °C for 10 min in buffer A. The resulting solution was diluted by 100-fold (i.e. 1 nM complex) with buffer A containing the oxygen scavenger system (25 mM glucose, 2.5 µM glucose oxidase, 10 nM catalase, and 10 mM dithiothreitol) and flowed into a flow cell made from a glass slide and coverslip for the successive fluorescent observation at room temperature.
The positions of individual 488-TkCPN and Cy5-PhPFD molecules adsorbed on the glass slide were visualized by total internal reflection fluorescence microscopy (TIRFM) (30). 488-TkCPN molecules were illuminated with a semiconductor laser (6.0 milliwatts, 473 nm, model ML0250A; Nippon Avionic Co. Inc., Tokyo, Japan). Cy5-PhPFD molecules were illuminated with a He-Ne laser (2.8 milliwatts, 632.8 nm, model GLG5350; NEC Co., Tokyo, Japan). The fluorescence emission from the specimen was collected with an oil-immersion microscope objective (1.40 numerical objective, x100, PlanApo; Olympus, Tokyo, Japan). Images were taken by a silicon-intensified tube camera (C2400–08, Hamamatsu Photonics, Shizuoka, Japan) coupled to an image intensifier (VS4-1845, Video Scope International) and recorded on videotapes for subsequent analysis. At least two fields of images were recorded for each assay, and statistical analysis was made from five independent assays. The positions of 488-TkCPN and Cy5-PhPFD at the same fields were marked individually using Scion Image software (Scion Corp.).
Surface Plasmon Resonance Detection of Prefoldin-Chaperonin Interaction—The surface plasmon resonance experiments were performed with a Biacore J biosensor system (Biacore AB, Uppsala, Sweden) at the sensor temperature of 25 °C. PhPFD or PhCPN was coupled to the sensor chip (CM5 research grade) via standard N-hydroxysuccinimide and N-ethyl-N-(dimethylaminopropyl)carbodiimide activation. For immobilizing chaperonins, 190 µl of 50 µg/ml protein in 10 mM sodium acetate (pH 5.0) was injected on the sensor surface. 190 µl of 6 µg/ml prefoldin in 10 mM potassium phosphate buffer (pH 7.5) was injected on the sensor surface to immobilize prefoldin. Ethanolamine was then injected to quench the unreacted N-hydroxysuccinimide groups. The mobile phase buffer used was HBS-EP buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.005% surfactant P-20). Analytes were injected at various concentrations, and the bound analytes were removed by washing with buffer 180 s after the injection.
Kinetic Analysis of Sensorgram Data—Kinetic constants were calculated from the sensorgram with BIAevaluation software, version 3.1 (Biacore), according to the global fitting model. The response curves for various analyte concentrations were globally fitted to several binding models provided with the above software. Apparent rate constants (kon1 and koff1 for the first step; kon2 and koff2 for the second step) were calculated based on the best fitted model, the bivalent model (first step: L + A 
L · A; second step: L · A + L L2 · A (L: ligand, A: analyte)). Dissociation constants (KD) were calculated by the resonance unit at equilibrium using the equation, Req = Rmax · C/(C + KD), where Req is equilibrium resonance units, Rmax is the resonance signal at saturation, and C is the concentration of free analyte. By dividing the KD values by that for the wild-type PhPFD, relative KD values were calculated.
Thermal Aggregation Measurements of CS—Thermal aggregation of CS from porcine heart was monitored by measuring the light scattering at 500 nm with a spectrofluorophotometer (RF-5300PC, Shimadzu, Kyoto, Japan) at 50 °C with continuous stirring. Monitoring started after addition of CS (80 nM as a monomer) to 50 mM Tris-HCl buffer, pH 8.0, with or without 240 nM of wild-type or mutant PhPFD complexes preincubated at 50 °C. Activity for protecting CS from thermal aggregation was defined as the relative reduction of light scattering by the presence of PhPFD complexes after 200 s.
Fluorometric Monitoring of GFP Refolding—The refolding of GFP denatured in 12.5 mM HCl was monitored by the fluorescence at 510 nm with excitation at 396 nm using a spectrofluorophotometer (RF-5300PC). Fluorescent measurements were initiated with the addition of 15 µM native or acid-denatured GFP (10 µl) into 1.5 ml of dilution buffer (50 mM Tris-HCl, pH 8.0, 100 mM KCl, 5 mM dithiothreitol, and 25 mM MgCl2) at 60 °C and under continuous stirring. To observe the chaperone activities, PhPFD complexes were added in the dilution buffer at a molar ratio of 1:5 (denatured GFP:prefoldin). Activity for arresting GFP refolding was defined as the relative reduction of fluorescence by the presence of PhPFD complexes after 600 s.
Other Methods—Proteins were analyzed by polyacrylamide gel electrophoresis on polyacrylamide gels containing SDS (SDS-PAGE) or polyacrylamide gels without SDS (Native-PAGE). Gels were stained with Coomassie Brilliant Blue R-250. Protein concentrations were measured by the method of Bradford with bovine serum albumin as the standard (31).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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chaperonin from Thermococcus sp. strain KS-1 (TkCPN) was used in this experiment, because we previously determined its crystal structure (15), which enabled us to select an appropriate position for labeling with a fluorescent dye (Alexa488). Equimolar amounts (100 nM) of fluorescently labeled TkCPN (488-TkCPN) and Cy5-labeled PhPFD (Cy5-PhPFD) were incubated at 60 °C, where both protein complexes are stable, and their individual signals were observed on a slide glass using a total internal reflection fluorescence microscopy (TIRFM) system (30) (Fig. 2). The location of TkCPN was identified by the fluorescence of Alexa488 and that of PhPFD by the fluorescence of Cy5, respectively. Approximately 30% of the Alexa488 spots were found to be superimposed on Cy5 spots, directly indicating that a significant fraction of the two chaperones formed binary complexes in the absence of substrate. Our findings indicate that, as with the eukaryotic prefoldin-chaperonin system (24, 32), the archaeal counterparts likely cooperate through direct interactions to facilitate the folding of non-native polypeptides.
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200 s after the removal of PhPFD. When PhPFD was immobilized, the signal also augmented when an increasing concentration of PhCPN was added to the mobile phase (Fig. 3B). The release of PhCPN was slow, however, and a considerable amount of PhCPN remained on the sensor chip even 200 s after the wash with buffer (Fig. 3B).
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d values of 23.3 for immobilized PhCPN and 6.99 for immobilized PhPFD, respectively, which were optimal compared with all other tested models. Apparent association and dissociation rate constants obtained from the fitted data models are shown in Table I. The rate constants for the first step (L + A L · A) for both experiments using PhPFD and PhCPN immobilized sensor chips are close to each other, but not identical. The discrepancy may be explained at least in part by the difference between the fitted model and real interaction mechanism and requires further investigation to obtain the actual kinetic constants for the interaction between PhPFD and PhCPN. The dissociation constant (KD), obtained using the data from immobilized PhCPN, was calculated to be 26.4 nM by the resonance unit at equilibrium. Although the conditions of binding may not reflect the proper physiological environment in which the interaction takes place (e.g. temperature among other factors), the value for the dissociation constant is suggestive of a particularly tight association between the two chaperones. This tight interaction is consistent with our above immunoprecipitation and single molecule observation results.
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10-fold reduced. In addition, a group I chaperonin from Thermus thermophilus HB8 exhibited little or no detectable interaction with PhPFD (Fig. 4A). Importantly, the affinity of Mt-PFD for PhCPN was also found to be highly reduced compared with that of PhPFD for the same chaperonin (Fig. 4B). Together, these data validate our above finding of a bona fide, specific interaction between PhPFD and PhCPN and suggest that the chaperone interactions are species-specific.
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subunits with a 5- or 6-amino acid truncation from the C terminus (tc5 or tc6, respectively) had almost the same ability to interact with the two non-native substrates as the wild-type (Table II). The effect of an 8-amino acid truncation from the C termini of the and subunits (tc8 and tc8, respectively) were then examined (Fig. 5). The PhPFD complex containing mutant subunit (tc8) failed to efficiently suppress the thermal aggregation of CS and had little ability to prevent GFP refolding (Fig. 5, B and C). On the other hand, the truncated subunit showed no observable effect on CS aggregation or GFP refolding (Fig. 5, B and C). Given that the removal of 5 or 6 residues from the C terminus of the PhPFD subunit had little effect but deletion of 8 residues had a considerable effect on the chaperone activity of the complex, we conclude that Leu111 and Ala110 of the last 8 amino acids (Ala110-Leu111-Arg112-Pro113-Pro114-Thr115-Ala116-Gly117-COOH) are important for chaperone-substrate interactions. Consistent with this result, amino acid replacement of Leu111 to Ala partially affects substrate binding (Table II). Importantly, residues from the N terminus of the subunit were also found to be important for substrate binding as a prefoldin complex containing a 10-residue truncation in the subunit (wttn10) was found to have compromised chaperone activity (Table II).
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subunit to chaperone activity was found to be important but somewhat less critical. The 8-amino acid C-terminal truncation of subunit (tc8) had almost no effect on the molecular chaperone activities of the PhPFD complexes with wild or truncated subunits. However, a complex containing a 17-amino acid N-terminal truncation from the subunit (tn17) affected the ability of prefoldin to prevent the aggregation of CS, while not having an observable effect on GFP refolding.
Effects of PhPFD Truncations on the Interaction with Ph-CPN—From a recent electron microscopy image of eukaryotic prefoldin bound to the CCT chaperonin, presently unidentified distal regions within (one or more) "tentacles" of prefoldin are likely to contact the chaperonin cylinder (24). The recombinant subunits bearing truncations therefore allowed us to test which of the one or more regions within the coiled-coils of archaeal prefoldin interact with the chaperonin. We examined the interaction of the truncated mutants with PhCPN by surface plasmon resonance (SPR) using the chaperonin-bound sensor chip. A PhPFD complex lacking 8 C-terminal residues within the subunit (tc8wt) exhibited almost the same binding response as that of the wild-type complex (Fig. 6A). Importantly, analysis of a prefoldin complex lacking 8 C-terminal residues in the subunit revealed that this distal end region was critical for the interaction with PhCPN (Fig. 6, B and C).
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subunit (Table III). A mutant complex, wttc5, has nearly the same affinity for PhCPN, but that of wttc7 is decreased 10-fold. Remarkably, the residue Leu111 was found to be critical for the interaction of PhPFD with Ph-CPN, because its mutagenesis to alanine resulted in a similar decrease of affinity compared with the deletion of 8 C-terminal amino acids. The N-terminal region of the subunit was also found to be important for chaperonin interactions, because a 10-amino acid deletion (wttn10) showed substantially reduced binding to the chaperonin (15-fold higher relative KD). On the other hand, a prefoldin complex containing the wild-type subunit and 8- or 17-amino acid truncation in the C- or N-terminal regions of the subunit (tc8wt and tn17wt, respectively) had little effect on its interaction with chaperonin. Given the possible co-axial binding of multiple prefoldin tentacles to the apical regions of the chaperonin toroid (24), we tested the effect of combined and subunit truncations on chaperonin binding. Truncations of the subunit in the tc8 background, i.e. tc8tc(5, 6, 7 or 8), revealed an additive loss of affinity for the chaperonin when compared with the tc8 mutant subunit by itself (Table III). These data strongly suggest that both subunits bind the chaperonin cooperatively.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The kinetics of the reaction between prefoldin and chaperonin was studied using SPR. Simulated curves of the bivalent analyte model were best fit to the experimental curves (Fig. 3, A and B). It is thought that prefoldin and chaperonin interact through six coiled-coils of prefoldin and eight apical domains of the chaperonin. Thus, it is reasonable that the interaction is most optimally fitted by the multivalent model. The kinetic constants calculated from the sensorgrams with PhCPN- and PhPFD-immobilized sensor chips are different. It might be partly due to the irreversibility of the sensorgram obtained with the PhPFD-immobilized chip, which is likely to be caused by the binding of PhCPN to multiple PhPFDs on a chip.
In this study, we obtained evidence for a species-specific interaction between prefoldin and its cognate chaperonin. Despite the high sequence homology between TkCPN and PhCPN, their affinities for PhPFD were considerably different. Although we demonstrated cooperation between PhPFD and Tth-CPN in the previous paper, we could not observe significant interaction between PhPFD and TthCPN by SPR. Recently, we have found that the binding between PhPFD and substrate is in dynamic equilibrium.3 Thus, it might be that the substrate was not directly transferred to TthCPN, but that protein released from PhPFD was captured by TthCPN.
Both N- and C-terminal ends of subunit are critical for interaction with unfolded proteins and chaperonins. In particular, the binding site in the C-terminal region was localized to the C-terminal 8 amino acid residues of subunit (Ala110-Leu111-Arg112-Pro113-Pro114-Thr115-Ala116-Gly117-COOH). Specifically, Ala110 and Leu111 appear to be essential for the interaction of prefoldin with unfolded proteins. Although we show that the hydrophobicity of Leu111 is important for the interaction, its contribution is only marginal (Table II). For the interaction with PhCPN, KD gradually increased with the deletion from 5 to 7 amino acids. Thus, the binding site for the chaperonin is adjacent to that of an unfolded protein, which may correlate with the effective handover of an aggregation-prone polypeptide to a chaperonin. The main difference between substrate recognition and chaperonin binding is the lowered affinity for chaperonin of tc8tc5, which exhibits almost same affinities for CS and GFP as that of the wild-type. We have observed acceleration of release of substrate from the prefoldin by chaperonins.3 The effect was not observed for the mutant PhPFD complex, tc8tc5t, which correlates with the decrease of the affinity with the chaperonin. Therefore, there is likely to be cooperation between and subunits in the interaction with a chaperonin, which is important for the substrate handover to the chaperonin.
A ternary complex of the prefoldin, a chaperonin, and a substrate protein should occur transiently during the substrate handover from the prefoldin and chaperonin. Thus, it is reasonable that the interaction between the prefoldin and the chaperonin is affected by the presence of a substrate protein. Contrary to our expectation, almost no significant difference was observed in the immunoprecipitation experiment between the presence and absence of denatured proteins (Fig. 1). To elucidate substrate handover mechanism, the affect of a substrate protein on the kinetics for the interaction between the prefoldin and chaperonin should be examined in more detail.
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FOOTNOTES |
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Both authors contributed equally to this work.
Supported by the Canadian Institutes of Health Research (CIHR) and the Michael Smith Foundation for Health Research scholarship awards.
To whom correspondence should be addressed. Tel./Fax: 81-42-388-7479; E-mail: yohda{at}cc.tuat.ac.jp.
1 The abbreviations used are: CCT, cytosolic chaperonin containing TCP-1; PFD, prefoldin; PhCPN, chaperonin from P. horikoshii OT3; PhPFD, prefoldin from P. horikoshii OT3; MtPFD, prefoldin from M. thermoautotrophicum; GFP, green fluorescent protein; CS, citrate synthase from porcine heart; SPR, surface plasmon resonance; TkCPN, chaperonin from Thermococcus sp. strain KS-1; TthCPN, chaperonin from T. thermophilus HB8; wt, wild-type PhPFD subunit; wt, wild-type PhPFD subunit; TIRFM, total internal reflection fluorescence microscopy; RU, resonance unit(s).
2 M. Okochi, H. Matsuzaki, T. Nomura, N. Ishii, and M. Yohda, manuscript in preparation.
3 T. Zako, R. Iizuka, M. Okochi, T. Ueno, H. Tadakuma, M. Yohda, and T. Funatsu, manuscript in preparation.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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