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• W1982408965 abstract "The Escherichia coli chaperonin GroEL is a double-ring chaperone that assists in protein folding with the aid of GroES and ATP. It is believed that GroEL alternates the folding-active rings and that the substrate protein (and GroES) can bind to the open trans-ring only after ATP in the cis-ring is hydrolyzed. However, we found that a substrate protein prebound to the trans-ring remained bound during the first ATP cycle, and this substrate was assisted by GroEL-GroES when the second cycle began. Moreover, a slow ATP-hydrolyzing GroEL mutant (D398A) in the ATP-bound form bound a substrate protein and GroES to the trans-ring. The apparent discrepancy with the results from an earlier study (Rye, H. S., Roseman, A. M., Chen, S., Furtak, K., Fenton, W. A., Saibil, H. R., and Horwich, A. L. (1999) Cell 97, 325–338) can be explained by the previously unnoticed fact that the ATP-bound form of the D398A mutant exists as a symmetric 1:2 GroEL-GroES complex (the “football”-shaped complex) and that the substrate protein (and GroES) in the medium is incorporated into the complex only after the slow turnover. In light of these results, the current model of the GroEL-GroES reaction cycle via the asymmetric 1:1 GroEL-GroES complex deserves reexamination. The Escherichia coli chaperonin GroEL is a double-ring chaperone that assists in protein folding with the aid of GroES and ATP. It is believed that GroEL alternates the folding-active rings and that the substrate protein (and GroES) can bind to the open trans-ring only after ATP in the cis-ring is hydrolyzed. However, we found that a substrate protein prebound to the trans-ring remained bound during the first ATP cycle, and this substrate was assisted by GroEL-GroES when the second cycle began. Moreover, a slow ATP-hydrolyzing GroEL mutant (D398A) in the ATP-bound form bound a substrate protein and GroES to the trans-ring. The apparent discrepancy with the results from an earlier study (Rye, H. S., Roseman, A. M., Chen, S., Furtak, K., Fenton, W. A., Saibil, H. R., and Horwich, A. L. (1999) Cell 97, 325–338) can be explained by the previously unnoticed fact that the ATP-bound form of the D398A mutant exists as a symmetric 1:2 GroEL-GroES complex (the “football”-shaped complex) and that the substrate protein (and GroES) in the medium is incorporated into the complex only after the slow turnover. In light of these results, the current model of the GroEL-GroES reaction cycle via the asymmetric 1:1 GroEL-GroES complex deserves reexamination. Chaperonins are a conserved class of molecular chaperones that assist in protein folding in the cell and are found in eubacteria, mitochondria, chloroplasts, archaea, and the eukaryotic cytosol (1Hartl F.U. Hayer-Hartl M. Science. 2002; 295: 1852-1858Crossref PubMed Scopus (2799) Google Scholar, 2Young J.C. Agashe V.R. Siegers K. Hartl F.U. Nat. Rev. Mol. Cell Biol. 2004; 5: 781-791Crossref PubMed Scopus (944) Google Scholar). The best characterized of these is the Escherichia coli chaperonin GroEL and its partner, GroES (3Thirumalai D. Lorimer G.H. Annu. Rev. Biophys. Biomol. Struct. 2001; 30: 245-269Crossref PubMed Scopus (330) Google Scholar, 4Taguchi H. J. Biochem. (Tokyo). 2005; 137: 543-549Crossref PubMed Scopus (32) Google Scholar, 5Horwich A.L. Fenton W.A. Chapman E. Farr G.W. Annu. Rev. Cell Dev. Biol. 2007; 23: 115-145Crossref PubMed Scopus (344) Google Scholar). Because GroEL is known to assist in the folding of hundreds of proteins, including those that are essential for cell growth (6McLennan N. Masters M. Nature. 1998; 392: 139Crossref PubMed Scopus (89) Google Scholar, 7Kerner M.J. Naylor D.J. Ishihama Y. Maier T. Chang H.C. Stines A.P. Georgopoulos C. Frishman D. Hayer-Hartl M. Mann M. Hartl F.U. Cell. 2005; 122: 209-220Abstract Full Text Full Text PDF PubMed Scopus (518) Google Scholar, 8Fujiwara K. Taguchi H. J. Bacteriol. 2007; 189: 5860-5866Crossref PubMed Scopus (35) Google Scholar), the molecular mechanism by which GroEL and GroES facilitate protein folding has been a key issue in the fields of chaperones and protein folding for almost 2 decades. GroEL is a large cylindrical protein complex comprising two heptameric rings of identical 57-kDa subunits, and these rings are stacked back to back (9Braig K. Otwinowski Z. Hegde R. Boisvert D.C. Joachimiak A. Horwich A.L. Sigler P.B. Nature. 1994; 371: 578-586Crossref PubMed Scopus (1194) Google Scholar, 10Xu Z. Horwich A.L. Sigler P.B. Nature. 1997; 388: 741-750Crossref PubMed Scopus (1044) Google Scholar). GroES is a single heptameric ring of identical 10-kDa subunits (11Hunt J.F. Weaver A.J. Landry S.J. Gierasch L. Deisenhofer J. Nature. 1996; 379: 37-45Crossref PubMed Scopus (404) Google Scholar). A large conformational change induced by ATP binding to GroEL promotes the formation of the GroEL-GroES complex (10Xu Z. Horwich A.L. Sigler P.B. Nature. 1997; 388: 741-750Crossref PubMed Scopus (1044) Google Scholar). The GroEL ring that binds ATP and GroES is called the cis-ring, and it has a cavity for the encapsulation of the substrate protein (the cis-cavity) (12Weissman J.S. Hohl C.M. Kovalenko O. Kashi Y. Chen S. Braig K. Saibil H.R. Fenton W.A. Horwich A.L. Cell. 1995; 83: 577-587Abstract Full Text PDF PubMed Scopus (391) Google Scholar, 13Mayhew M. da Silva A.C. Martin J. Erdjument-Bromage H. Tempst P. Hartl F.U. Nature. 1996; 379: 420-426Crossref PubMed Scopus (345) Google Scholar). The origin of the asymmetry in the GroEL-GroES complex is explained by the nested cooperativity in the ATP binding, the positive cooperativity within the same GroEL rings, and the negative cooperativity between the two rings (14Horovitz A. Willison K.R. Curr. Opin. Struct. Biol. 2005; 15: 646-651Crossref PubMed Scopus (126) Google Scholar). The most efficient manner of GroEL-GroES-assisted folding involves the substrate protein (up to ∼57 kDa (15Sakikawa C. Taguchi H. Makino Y. Yoshida M. J. Biol. Chem. 1999; 274: 21251-21256Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar)) bound to GroEL being ejected into the cis-cavity upon the ATP-dependent formation of the GroEL-GroES complex. ATP hydrolysis in the cis-ring results in the formation of the asymmetric ADP-bound GroEL-GroES complex (called the “ADP bullet”). Subsequent ATP binding to the opposite side of the GroEL ring (the trans-ring) induces the release of GroES, ADP, and the encapsulated substrate (either folded or not (16Todd M.J. Viitanen P.V. Lorimer G.H. Science. 1994; 265: 659-666Crossref PubMed Scopus (429) Google Scholar, 17Weissman J.S. Kashi Y. Fenton W.A. Horwich A.L. Cell. 1994; 78: 693-702Abstract Full Text PDF PubMed Scopus (331) Google Scholar, 18Taguchi H. Yoshida M. FEBS Lett. 1995; 359: 195-198Crossref PubMed Scopus (34) Google Scholar)) from the cis-ring (19Rye H.S. Burston S.G. Fenton W.A. Beechem J.M. Xu Z. Sigler P.B. Horwich A.L. Nature. 1997; 388: 792-798Crossref PubMed Scopus (357) Google Scholar, 20Rye H.S. Roseman A.M. Chen S. Furtak K. Fenton W.A. Saibil H.R. Horwich A.L. Cell. 1999; 97: 325-338Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar). The ATP-bound trans-ring is then reoriented to a new cis-ring, thereby allowing the next ATPase cycle. Multiple rounds of the GroEL cycle are required for the productive folding of stringent substrates, such as rhodanese and ribulose-bisphosphate carboxylase/oxygenase (Rubisco) 3The abbreviations used are:Rubiscoribulose-bisphosphate carboxylase/oxygenaseHPLChigh pressure liquid chromatographyBeFxfluoroberyllate. (16Todd M.J. Viitanen P.V. Lorimer G.H. Science. 1994; 265: 659-666Crossref PubMed Scopus (429) Google Scholar, 17Weissman J.S. Kashi Y. Fenton W.A. Horwich A.L. Cell. 1994; 78: 693-702Abstract Full Text PDF PubMed Scopus (331) Google Scholar, 18Taguchi H. Yoshida M. FEBS Lett. 1995; 359: 195-198Crossref PubMed Scopus (34) Google Scholar). ribulose-bisphosphate carboxylase/oxygenase high pressure liquid chromatography fluoroberyllate. The key experiments to establish the above alternation model were a series of studies using an ATPase-deficient GroEL mutant in which Asp398 was replaced with Ala (EL398) (19Rye H.S. Burston S.G. Fenton W.A. Beechem J.M. Xu Z. Sigler P.B. Horwich A.L. Nature. 1997; 388: 792-798Crossref PubMed Scopus (357) Google Scholar, 20Rye H.S. Roseman A.M. Chen S. Furtak K. Fenton W.A. Saibil H.R. Horwich A.L. Cell. 1999; 97: 325-338Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar). EL398 is deficient in ATP hydrolysis (<2% of the wild type) but not in ATP binding and thus forms a long-lived ATP-bound GroEL-GroES complex (asymmetric GroEL-GroES complex, called the ATP bullet). Rye et al. (20Rye H.S. Roseman A.M. Chen S. Furtak K. Fenton W.A. Saibil H.R. Horwich A.L. Cell. 1999; 97: 325-338Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar) reported that the trans-ring of the ATP bullet cannot bind either GroES or the substrate protein. ATP hydrolysis in the cis-ring of the ATP bullet permits the binding of both ATP and the substrate to the trans-ring (19Rye H.S. Burston S.G. Fenton W.A. Beechem J.M. Xu Z. Sigler P.B. Horwich A.L. Nature. 1997; 388: 792-798Crossref PubMed Scopus (357) Google Scholar, 20Rye H.S. Roseman A.M. Chen S. Furtak K. Fenton W.A. Saibil H.R. Horwich A.L. Cell. 1999; 97: 325-338Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar). Substrates can bind to both of the GroEL rings at the same time (a substrate-saturated GroEL) (21Sparrer H. Rutkat K. Buchner J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1096-1100Crossref PubMed Scopus (73) Google Scholar, 22Motojima F. Yoshida M. J. Biol. Chem. 2003; 278: 26648-26654Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 23Taguchi H. Tsukuda K. Motojima F. Koike-Takeshita A. Yoshida M. J. Biol. Chem. 2004; 279: 45737-45743Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 24Koike-Takeshita A. Shimamura T. Yokoyama K. Yoshida M. Taguchi H. J. Biol. Chem. 2006; 281: 962-967Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). The availability of the substrate-saturated GroEL prompted us to examine whether the ATP bullet, formed from the substrate-saturated GroEL, retains the bound substrate in the trans-ring because the previous experiment using EL398 started from the GroEL mutant without bound substrates. In this study, we addressed this question and found that the bound substrate in the trans-ring remained bound during the GroEL cycle. In addition, we reevaluated the mechanism of EL398 and unexpectedly found a discrepancy between our results and those from a previous study (20Rye H.S. Roseman A.M. Chen S. Furtak K. Fenton W.A. Saibil H.R. Horwich A.L. Cell. 1999; 97: 325-338Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar). We resolved the apparent discrepancy by a careful reexamination of EL398. Revisiting the GroEL cycle model implied an efficient two-stroke mechanism (25Lorimer G. Nature. 1997; 388: 720-721Crossref PubMed Scopus (79) Google Scholar) via the symmetric football complex. Proteins and Reagents—Hexokinase was from Sigma. Proteinase K, ATP, and ADP were obtained from Roche Applied Science. The trace amount of contaminating ATP in the ADP solution was eliminated by a hexokinase/glucose treatment (22Motojima F. Yoshida M. J. Biol. Chem. 2003; 278: 26648-26654Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Cy3-N-hydroxysuccinimide (FluoroLink Cy3 monofunctional dye) was from GE Healthcare. The following proteins were purified and prepared as described previously: wild-type and mutant GroEL, GroES, and bovine mitochondrial rhodanese (26Motojima F. Makio T. Aoki K. Makino Y. Kuwajima K. Yoshida M. Biochem. Biophys. Res. Commun. 2000; 267: 842-849Crossref PubMed Scopus (39) Google Scholar); Rubisco from Rhodospirillum rubrum (19Rye H.S. Burston S.G. Fenton W.A. Beechem J.M. Xu Z. Sigler P.B. Horwich A.L. Nature. 1997; 388: 792-798Crossref PubMed Scopus (357) Google Scholar); and Cy3-labeled GroES (Cy3-GroES) and Cy3-labeled substrate (Cy3-rhodanese and Cy3-Rubisco) (27Taguchi H. Ueno T. Tadakuma H. Yoshida M. Funatsu T. Nat. Biotechnol. 2001; 19: 861-865Crossref PubMed Scopus (102) Google Scholar). SDS-PAGE Analysis of GroEL-GroES-Substrate Ternary Complexes—GroEL and EL398, which had been saturated with denatured rhodanese, were prepared as described previously (22Motojima F. Yoshida M. J. Biol. Chem. 2003; 278: 26648-26654Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Briefly, rhodanese was heat-denatured at 60 °C for 15 min in HKM buffer (20 mm HEPES-KOH (pH 7.4), 100 mm KCl, and 5 mm MgCl2) containing GroEL. To initiate the GroEL ATPase cycle, the solution containing GroEL (or EL398), which was saturated with rhodanese, and GroES in HKM buffer was mixed with a 2-fold volume of the solution containing ATP and then incubated at 25 °C. The final concentrations of the components in the reaction mixtures were as follows: 1 mm ATP, 0.5 μm GroEL or EL398 saturated with rhodanese, 1.0 μm GroES, 200 mm glucose, 1 mm dithiothreitol, and 20 mm Na2S2O3. For the single turnover ATP hydrolysis experiment (denoted “ATPsingle”), the excess ATP was hydrolyzed to ADP by adding hexokinase (final concentration, 0.04 units/μl) to the reaction mixture at 3 s after the initiation of the reaction (22Motojima F. Yoshida M. J. Biol. Chem. 2003; 278: 26648-26654Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). To determine the amounts of GroES and rhodanese bound to GroEL or EL398, the released GroES and rhodanese were removed by ultrafiltration (Microcon YM-100, Millipore). Subsequently, proteinase K (final concentration, 1 μg/ml) was added to an aliquot of the solution. Following an incubation for 30 min at 25 °C, the components with molecular masses <100 kDa were removed by ultrafiltration (Microcon YM-100). The retained solution was analyzed by 13% SDS-PAGE. The intensity of the band staining was quantified using the ImageJ program and was calibrated using known protein concentrations. To quantify the amount of GroES bound to EL398, EL398-GroES complexes formed in the presence of ATP, ADP, ATP with 10 mm NaF and 1 mm BeCl2, or ADP with 10 mm NaF and 1 mm BeCl2 were also analyzed by 13% SDS-PAGE as described above. Rhodanese Folding Assay—The folding assays were conducted in HKM buffer including 200 mm glucose, 1 mm dithiothreitol, and 20 mm Na2S2O3. The final concentrations were 0.5 μm GroEL or EL398 saturated with rhodanese and 1.5 μm GroES. Folding was initiated by the addition of either ATP or ADP at a final concentration of 1 mm. For the single turnover ATP hydrolysis reactions, the excess ATP was removed by adding hexokinase (final concentration, 0.04 units/μl) to the reaction mixture at 3 s after the initiation of the reaction. For the repeated single turnover experiment (see Fig. 1B), additional ATP (final concentration, 5 mm) was mixed in the reaction mixture at 25 min after the initiation of the first single turnover experiment. At the times indicated, the folding was quenched by mixing aliquots of the solution with 750 μl of a solution containing 100 mm KH2PO4, 150 mm Na2S2O3, and 1 mm EDTA. After the quench, the enzymatic assay was initiated by adding 250 μl of 0.25 m KCN at 25 °C and was stopped by adding 200 μl of 37% formaldehyde after 15 min. After the further addition of 0.5 ml of ferric nitrate reagent (100 g of Fe(NO3)3·9H2O and 200 ml of HNO3, brought to 1000 ml with H2O), the rhodanese activity was measured colorimetrically by the absorbance at 460 nm (28Sörbo B.H. Acta Chem. Scand. 1953; 7: 1129-1136Crossref Google Scholar), indicating the formation of a complex between the ferric ions and the thiocyanate reaction product. Binding Assay Using Gel Filtration—The ATP bullet and the ATP football complexes of EL398 were formed by mixing 1.5 μm EL398 with 1.5 μm and 3 μm GroES, respectively, 1 mm dithiothreitol, and 1 mm ATP in HKM buffer to initiate the ATP hydrolysis reaction (t = 0). Following an incubation for 30 s at 25 °C, the complexes were isolated by gel filtration (PD-10 column, GE Healthcare) in the same buffer. The capacity of binding of the complexes or unliganded EL398 to GroES was then examined by mixing 0.5 μm complexes or unliganded EL398 with 1 μm Cy3-GroES in 1 mm ATP at the indicated times following the initiation of ATP hydrolysis. The capacity of binding of the complexes or unliganded EL398 to denatured substrates was examined by mixing 0.5 μm complexes or unliganded EL398 with either 0.25 μm urea-denatured Cy3-rhodanese or 0.25 μm acid-denatured Cy3-Rubisco at the indicated times following the initiation. After a 5-min incubation at 25 °C, the samples were analyzed with a gel filtration HPLC column (G3000SWXL, Tosoh Corp.). Aliquots were loaded onto the column, which was equilibrated with HKM buffer containing 50 mm Na2SO4. The flow rate was 0.5 ml/min, and the elution profile was monitored for the Cy3 fluorescence using an in-line fluorometer (excitation at 550 nm and emission at 570 nm). Quantification of Bound Nucleotides—To analyze the bound nucleotides in the EL398-GroES complex, we mixed 2 μm EL398, 4 μm GroES, 1 mm dithiothreitol, 1 mm ATP, 20 mm HEPES-KOH (pH 7.4), 50 mm KCl, and 5 mm MgCl2. After 5 min, aliquots were rapidly subjected to gel filtration using three TSK-GEL guard columns (Tosoh Corp.) connected in series and equilibrated with buffer containing 25 mm HEPES-KOH (pH 7.0), 100 mm Na2SO4, and 5 mm MgSO4. The isolated EL398 complexes were treated with perchloric acid (final concentration, 1.0%), and the supernatant was neutralized with K2CO3. The supernatants were applied to a reverse-phase HPLC column (ODS-80Ts, Tosoh Corp.) for the separation of ATP and ADP by monitoring the absorbance at 260 nm (29Hisabori T. Muneyuki E. Odaka M. Yokoyama K. Mochizuki K. Yoshida M. J. Biol. Chem. 1992; 267: 4551-4556Abstract Full Text PDF PubMed Google Scholar). The amount of nucleotide was calculated by the integrated peak area and was calibrated using known nucleotide concentrations. Denatured Rhodanese Bound to the trans-Ring Remains Bound during GroEL-GroES Cycling—We have developed a method to prepare substrate-saturated GroEL, in which the substrates are bound to both of the GroEL rings (22Motojima F. Yoshida M. J. Biol. Chem. 2003; 278: 26648-26654Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 23Taguchi H. Tsukuda K. Motojima F. Koike-Takeshita A. Yoshida M. J. Biol. Chem. 2004; 279: 45737-45743Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 24Koike-Takeshita A. Shimamura T. Yokoyama K. Yoshida M. Taguchi H. J. Biol. Chem. 2006; 281: 962-967Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). The addition of ATP and GroES to the substrate-saturated GroEL results in the formation of an ATP-bound asymmetric GroEL-GroES complex (22Motojima F. Yoshida M. J. Biol. Chem. 2003; 278: 26648-26654Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 23Taguchi H. Tsukuda K. Motojima F. Koike-Takeshita A. Yoshida M. J. Biol. Chem. 2004; 279: 45737-45743Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), leading to the encapsulation of one substrate bound to the cis-ring within the cis-cavity. What is the fate of the other substrate bound to the trans-ring? Although an earlier key experiment in which the ATP-bound asymmetric GroEL-GroES complex (ATP bullet) could not bind the substrate protein in the trans-ring (20Rye H.S. Roseman A.M. Chen S. Furtak K. Fenton W.A. Saibil H.R. Horwich A.L. Cell. 1999; 97: 325-338Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar) suggested that the other substrate in the trans-ring should be released, no experiments were performed to clarify this point. To address this question, we prepared the substrate-saturated GroEL. The saturation protocol reproducibly resulted in the binding of 2.5∼3 mol of denatured rhodanese/mol of GroEL tetradecamer (Fig. 1A) (22Motojima F. Yoshida M. J. Biol. Chem. 2003; 278: 26648-26654Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 23Taguchi H. Tsukuda K. Motojima F. Koike-Takeshita A. Yoshida M. J. Biol. Chem. 2004; 279: 45737-45743Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). We note that both of the rhodanese-bound GroEL rings efficiently bound GroES as evident from our previous studies (22Motojima F. Yoshida M. J. Biol. Chem. 2003; 278: 26648-26654Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 23Taguchi H. Tsukuda K. Motojima F. Koike-Takeshita A. Yoshida M. J. Biol. Chem. 2004; 279: 45737-45743Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). The rhodanese bound to GroEL was completely digested by proteinase K (Fig. 1A, lanes 1 and 2), indicating that the bound rhodanese was in a protease-susceptible state. ATP was then added in the presence of GroES to initiate the GroEL ATPase cycle. After the reaction proceeded for 3 s, hexokinase was added to the solution (containing glucose) to hydrolyze all of the free ATP to prevent a second GroEL ATPase cycle. After the single turnover experiment (referred to as ATPsingle (22Motojima F. Yoshida M. J. Biol. Chem. 2003; 278: 26648-26654Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar)), the GroEL complexes were treated with proteinase K to digest the exposed denatured rhodanese and then subjected to ultrafiltration (100-kDa cutoff membrane) to separate the digested/released rhodanese or GroES. Quantification of GroES based on the band densities revealed that 1.0 mol of GroES/mol of GroEL was retained in the isolated GroEL-GroES complex (Fig. 1A, lanes 3 and 4), confirming the efficient formation of the asymmetric GroEL-GroES complex in the single turnover experiment using the rhodanese-saturated GroEL. The release of bound rhodanese from the trans-ring, as predicted from the current GroEL model, should result in no difference in the band intensity of rhodanese with or without the protease treatment. However, we found that the 2.8 mol of rhodanese preloaded with the GroEL-GroES complex had been reduced to 1.2 mol after the protease treatment (Fig. 1A, lanes 3 and 4). Because 1.0 mol of GroES was retained in the isolated ternary complex, the protease-protected rhodanese is considered to have been encapsulated in the cis-cavity (22Motojima F. Yoshida M. J. Biol. Chem. 2003; 278: 26648-26654Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 23Taguchi H. Tsukuda K. Motojima F. Koike-Takeshita A. Yoshida M. J. Biol. Chem. 2004; 279: 45737-45743Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 24Koike-Takeshita A. Shimamura T. Yokoyama K. Yoshida M. Taguchi H. J. Biol. Chem. 2006; 281: 962-967Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Therefore, the presence of the protease-susceptible rhodanese would indicate the retention of the substrate by the trans-ring after the completion of the single turnover experiment. Next, we tested whether the folding of the trans-retained rhodanese is still assisted by GroEL-GroES. As shown in our previous studies (22Motojima F. Yoshida M. J. Biol. Chem. 2003; 278: 26648-26654Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 23Taguchi H. Tsukuda K. Motojima F. Koike-Takeshita A. Yoshida M. J. Biol. Chem. 2004; 279: 45737-45743Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), ∼2 mol of rhodanese/mol of GroEL tetradecamer was recovered when ATP and GroES were added to the rhodanese-saturated GroEL. In contrast, ADP and GroES did not assist in the folding of rhodanese. The ATPsingle condition resulted in a gradual recovery of the rhodanese activity until it reached a value corresponding to ∼0.8 mol of rhodanese/mol of GroEL (Fig. 1B), showing that the rhodanese encapsulated in the cis-cavity was folded under the ATPsingle condition as reported previously (22Motojima F. Yoshida M. J. Biol. Chem. 2003; 278: 26648-26654Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 23Taguchi H. Tsukuda K. Motojima F. Koike-Takeshita A. Yoshida M. J. Biol. Chem. 2004; 279: 45737-45743Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). At 25 min under the ATPsingle condition, ATP was added to the mixture to trigger a second GroEL cycle. The second addition of ATP re-induced the folding of rhodanese, reaching a value of ∼1.4 mol of rhodanese/mol of GroEL, which was almost twice the recovery compared with that before the addition of the second ATP. The second recovery of rhodanese was not inhibited by the inclusion of a trap-GroEL, which binds the substrate but does not release it even in the presence of ATP (12Weissman J.S. Hohl C.M. Kovalenko O. Kashi Y. Chen S. Braig K. Saibil H.R. Fenton W.A. Horwich A.L. Cell. 1995; 83: 577-587Abstract Full Text PDF PubMed Scopus (391) Google Scholar), in the solution (data not shown), indicating that the trans-bound substrate was not released into the bulk solution during ATP hydrolysis in the cis-ring. Therefore, we concluded that the trans-ring of the cis-ATP complex retains the substrate that is available for GroEL-GroES-dependent folding. The GroEL(D398A) Mutant Binds Two GroES Heptamers in the Presence of ATP, Producing a Symmetric “ATP Football” Complex—Because the ATPsingle condition experiment was conducted with wild-type GroEL, it was possible that the trans-bound substrate might have been transiently released from the ATP bullet but then bound again to the trans-ring after the formation of the ADP bullet as a result of ATP hydrolysis. To rule out this possibility, we conducted the above protease protection experiment using EL398 because this mutant remains in the ATP-bound form with a half-life of ∼30 min (19Rye H.S. Burston S.G. Fenton W.A. Beechem J.M. Xu Z. Sigler P.B. Horwich A.L. Nature. 1997; 388: 792-798Crossref PubMed Scopus (357) Google Scholar, 20Rye H.S. Roseman A.M. Chen S. Furtak K. Fenton W.A. Saibil H.R. Horwich A.L. Cell. 1999; 97: 325-338Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar), which is long enough to isolate the ATP-bound EL398-GroES complex in our protocol. The release of the trans-bound rhodanese by the ATP-bound EL398 complex should result in the isolation of a trans-free EL398-GroES complex after ultrafiltration. At first, we conducted the ATPsingle condition experiment using EL398 as a control (Fig. 2A). We expected that about half of the bound rhodanese would be digested by proteinase K because the ATPsingle condition should produce the asymmetric ATP bullet complex, which has protease-susceptible rhodanese in the trans-ring. However, we found only a slight reduction in the amount of rhodanese after the protease treatment (Fig. 2A, lanes 1 and 2). Quantification of the retained rhodanese revealed that 2.7 mol of rhodanese was protected after the protease treatment (Fig. 2A, lane 2). In addition, we noticed that ∼2 mol of GroES/mol of GroEL was bound to the EL398 complex after the completion of a single turnover (Fig. 2A, lanes 1 and 2), suggesting the formation of a symmetric 1:2 EL398-GroES complex (ATP football). Because the formation of the stable ATP football complex would result in the encapsulation of rhodanese in both cavities, we then compared the recovery of the rhodanese activity using EL398 with that using wild-type GroEL under the ATPsingle condition in the presence of GroES. Strikingly, the recovery of the rhodanese activity assisted by EL398-GroES was almost double that assisted by wild-type GroEL-GroES (Fig. 2B). This means that the EL398-GroES complex has two folding chambers to assist in the folding of ∼2 mol of rhodanese/mol of EL398 tetradecamer, further supporting the formation of the ATP football complex. We next examined whether the substrates are released by the ATP-bound EL398 complex. Separation of the EL398-GroES complex at 1 min after the addition of ATP resulted in the retention of ∼2 mol of the protease-protected rhodanese (Fig. 2A, lanes 3 and 4). In addition, ∼2 mol of GroES was retained. The retention of both cis- and trans-bound substrates was interpreted as an encapsulation of both substrates in two cis-cavities of the ATP football. The retention of rhodanese was reduced to <1 mol after 120 min (Fig. 2A, lanes 5 and 6). This reduction in the amount of retained rhodanese was interpreted as being the consequence of several rounds of EL398 ATPase turnover during the 120-min reaction, resulting in the release of native rhodanese from the EL398-GroES complex. Taken together, our experiments using EL398 strongly suggested the formation of the ATP football complex, which was not observed in previous studies using EL398 (19Rye H.S. Burston S.G. Fenton W.A. Beechem J.M. Xu Z. Sigler P.B. Horwich A.L. Nature. 1997; 388: 792-798Crossref PubMed Scopus (357) Google Scholar, 20Rye H.S. Roseman A.M. Chen S. Furtak K. Fenton W.A. Saibil H.R. Horwich A.L. Cell. 1999; 97: 325-338Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar). These unexpected results prompted us to carefully reevaluate EL398. Reevaluation of EL398: The Asymmetric ATP Bullet Complex Binds a Second GroES or Denatured Substrate to the trans-Ring—To compare the results reported by Rye et al. (20Rye H.S. Roseman A.M. Chen S. Furtak K. Fenton W.A. Saibil H.R. Horwich A.L. Cell. 1999; 97: 325-338Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar) with ours, EL398 without bound rhodanese was used. Because our previous experiment using fluoroberyllate (BeFx) revealed that the incubation of GroEL (wild-type) and GroES in the presence of ATP + BeFx and ADP + BeFx resulted in the formation of a stable football (Fig. 3A, lane 4) and a bullet (lane 5), respectively (23Taguchi H. Tsukuda K. Motojima F. Koike-Takeshita A. Yoshida M. J. Biol. Chem. 2004; 279: 45737-45743Abstract Full Text Full Text PD" @default.
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• W1982408965 title "Revisiting the GroEL-GroES Reaction Cycle via the Symmetric Intermediate Implied by Novel Aspects of the GroEL(D398A) Mutant" @default.
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