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Jbc.Org Interaction of Prefoldin with Group II Chaperonin 31789 THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 30, Issue of July 23, pp. 31788–31795, 2004 © 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Kinetics and Binding Sites for Interaction of the Prefoldin with a Group II Chaperonin CONTIGUOUS NON-NATIVE SUBSTRATE AND CHAPERONIN BINDING SITES IN THE ARCHAEAL PREFOLDIN* Received for publication, March 15, 2004, and in revised form, May 6, 2004 Published, JBC Papers in Press, May 15, 2004, DOI 10.1074/jbc.M402889200 Mina Okochi‡§, Tomoko Nomura‡§, Tamotsu Zako‡, Takatoshi Arakawa‡, Ryo Iizuka‡, Hiroshi Ueda¶, Takashi Funatsuʈ, Michel Leroux**‡‡, and Masafumi Yohda‡§§ 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 ʈDepartment 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 Prefoldin is a jellyfish-shaped hexameric co-chaper- in their central cavity to promote correct folding in an ATP-de- one of the group II chaperonins. It captures a protein pendent manner (2–5). They are classified into two groups, folding intermediate and transfers it to a group II chap- group I found in bacteria and organelles of eukaryotes, and eronin for completion of folding. The manner in which group II in archaea and in the cytoplasm of eukaryotes (6, 7). prefoldin interacts with its substrates and cooperates The bacterial group I chaperonin, GroEL, is a cylinder-shaped with the chaperonin is poorly understood. In this study, tetradecamer that is capped by the heptameric co-chaperone, we have examined the interaction between a prefoldin GroES (8–10). In contrast, the group II chaperonin exists as an and a chaperonin from hyperthermophilic archaea by eight- or nine-rotationally symmetric double ring in a toroidal immunoprecipitation, single molecule observation, and structure composed of homologous subunits of about 60 kDa surface plasmon resonance. We demonstrate that Pyro- and functions independently of a co-chaperone corresponding coccus prefoldin interacts most tightly with its cognate to GroES (11, 12). The crystal structures of the group II chap- chaperonin, and vice versa, suggesting species specific- eronins from the acidothermophilic archaeum Thermoplasma ity in the interaction. Using truncation mutants, we un- acidophilum and the hyperthermophilic archaeum Thermococ- covered by kinetic analyses that this interaction is mul- tivalent in nature, consistent with multiple binding sites cus sp. strain KS-1 suggest that the long helical protrusions between the two chaperones. We present evidence that located at the opening of their cavities, in their apical domains, both N- and C-terminal regions of the prefoldin ␤ sub- play the equivalent role of GroES as a built-in lid of the cavity unit are important for molecular chaperone activity and (13–15). The archaeal chaperonin takes an open conformation for the interaction with a chaperonin. Our data are con- in the nucleotide-free or ADP-bound states and changes to a sistent with substrate and chaperonin binding sites on closed conformation upon binding ATP (16, 17). Interestingly, prefoldin that are different but in close proximity, closure of the built-in lid of CCT,1 the eukaryotic cytosol group which suggests a possible handover mechanism of pre- II chaperonin, is induced not by the binding but by the hydrol- foldin substrates to the chaperonin. ysis of ATP (18). The group II chaperonins cooperate with a co-chaperone, prefoldin/GimC (19–21). Prefoldin (PFD) has been shown to Molecular chaperones are ubiquitous proteins that are re- participate in the maturation of actin and members of the quired for the correct folding, assembly, transport, and degra- tubulin family by transferring them in the incompletely folded dation of proteins within the cell (1). One class of chaperones, states to CCT. Although there is neither actin nor tubulin in termed chaperonins, are seven- to nine-membered double ring archaea, both prefoldin homologues and group II chaperonins complexes of 800–1000 kDa that capture non-native proteins 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 * This work was supported in part by grants-in-aid for scientific two different but related ␣ class subunits and four related ␤ research on priority areas (13033008, 14037216, and 15032212) and a class subunits. The crystal structure of the archaeal prefoldin grant of the National Project on Protein Structural and Functional from Methanobacterium thermoautotrophicum (MtPFD) has Analyses from the Ministry of Education, Science, Sports and Culture of Japan (to M. Y.). The work reported here is a part of the 21st Century been determined at a resolution of 2.3 Å (23). It resembles a COE (Center of Excellence) program of “Future Nano-Materials” re- jellyfish in that its body consists of a double ␤-barrel assembly search and education project, which is financially supported by the with six long tentacle-like coiled-coils protruding from it. The Ministry of Education, Science, Sports, Culture, and Technology through Tokyo University of Agriculture & Technology. The costs of publication of this article were defrayed in part by the payment of page 1 The abbreviations used are: CCT, cytosolic chaperonin containing charges. This article must therefore be hereby marked “advertisement” TCP-1; PFD, prefoldin; PhCPN, chaperonin from P. horikoshii OT3; in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. PhPFD, prefoldin from P. horikoshii OT3; MtPFD, prefoldin from M. § Both authors contributed equally to this work. thermoautotrophicum; GFP, green fluorescent protein; CS, citrate syn- ‡‡ Supported by the Canadian Institutes of Health Research (CIHR) thase from porcine heart; SPR, surface plasmon resonance; TkCPN, ␣ and the Michael Smith Foundation for Health Research scholarship chaperonin from Thermococcus sp. strain KS-1; TthCPN, chaperonin awards. from T. thermophilus HB8; wt␣, wild-type PhPFD ␣ subunit; wt␤, §§ To whom correspondence should be addressed. Tel./Fax: 81-42- wild-type PhPFD ␤ subunit; TIRFM, total internal reflection fluores- 388-7479; E-mail: [email protected]. cence microscopy; RU, resonance unit(s). 31788 This paper is available on line at http://www.jbc.org Interaction of Prefoldin with Group II Chaperonin 31789 distal regions of the coiled-coils partially expose hydrophobic (PH0532L111AFw 5Ј-GAT-TCA-AGC-CGC-TGC-GAG-GCC-GCC-TAC- Ј Ј patches, and there is evidence that coiled-coils are required for CGC-3 , PH0532L111ARv 5 -GCG-GTA-GGC-GGC-CTC-GCA-GCG- GCT-TGA-ATC-3Ј). The construct was also verified by DNA sequencing. the multivalent binding of non-native proteins (23). Eukaryotic Each subunit was expressed in E. coli BL21(DE3). After removal of prefoldin possesses a structure similar to that of archaeal pre- most E. coli proteins by heat treatment at 80 °C for 30 min, it was foldin and has been observed to capture a substrate protein purified by anion exchange chromatography on a DEAE-Toyopearl col- (actin) in the cavity formed by its six “tentacles” (24). In the umn (Tosoh, Tokyo, Japan) using buffer A (50 mM Tris-HCl, pH 8.0, 0.1 three-dimensional reconstitution of the prefoldin⅐actin com- mM EDTA) with a gradient of NaCl, and subsequent gel filtration on a plex, actin appears to interact with prefoldin in a region that is HiLoad 26/60 Superdex 200 prep grade column (Amersham Bio- sciences) equilibrated with buffer A containing 150 mM NaCl. PhPFD not well defined but is also consistent with the distal regions of complexes were reconstituted by incubating the mixture of the purified the coiled-coils (24). Additional evidence for actin and tubulin ␣ and ␤ subunits at the molar ratio of 1:2 at 80 °C for 30 min. The substrate interaction with the distal ends of different but over- reconstituted complex was purified by gel filtration on a Bio-Prep col- lapping sets of eukaryotic prefoldin subunits has been obtained umn (Bio-Rad). The purified complex contained ␣ and ␤ subunits at the recently (25). molar ratio of 1:2 as judged by SDS-PAGE analysis. In contrast, the interaction and functional cooperation be- Chaperonins from Pyrococcus horikoshii OT3 (PhCPN), Thermococ- cus sp strain KS-1 (TkCPN), and Thermus thermophilus HB8 (TthCPN) tween the archaeal chaperonin and prefoldin is comparably less were expressed and purified as described previously (17, 26).2 Wild-type understood. The archaeal chaperones represent a powerful model prefoldin from M. thermoautotrophicum, MtPFD, was prepared as de- system for examining their functional cooperation because of scribed previously (22). their simple subunit composition and structural stability. The GFP used in this report is a heat-stable mutant, with alanine We have already reported that the prefoldin from Pyrococcus inserted in the N-terminal region, a His tag in the C-terminal region, horikoshii OT3 (PhPFD) functionally cooperates with chaper- and amino acid substitution of F99S, M153T, V163A, and L165F (5, 27). It was purified as described previously (28). onins in the refolding of the green fluorescent protein (GFP) in Immunoprecipitation of Prefoldin with Anti-chaperonin Antibody— vitro (26). In the present study, we have examined the inter- The immunoprecipitation experiments were performed using IMMU- action between a prefoldin and a chaperonin from hyperther- NOcatcher (CytoSignal) to examine the interactions between the pre- mophilic archaea by immunoprecipitation, single molecule ob- foldin and the chaperonin in the presence or absence of denatured GFP. servation, and surface plasmon resonance, and characterized The anti-TkCPN antibody was used for the immunoprecipitation. Be- the substrate and chaperonin binding site of prefoldin.
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