Abstract
Chaperonins are oligomeric protein complexes that play an essential role in the cell, mediating ATP-dependent polypeptide chain folding in a variety of cellular compartments. They appear to bind early folding intermediates, preventing their aggregation; in the presence of MgATP and a cochaperonin, bound polypeptides are released in a stepwise manner, associated with folding to the native state. Chaperonin complexes appear in the electron microscope as cylindrical structures, usually composed of two stacked rings, each containing, by negative staining, an electron dense central "hole" approximately 6.0 nm in diameter. We sought to identify the site on the Escherichia coli chaperonin groEL, where the "molten globule"-like intermediate of dihydrofolate reductase (DHFR) becomes bound, by examining in the scanning transmission electron microscope complexes formed between groEL and DHFR molecules bearing covalently crosslinked 1.4-nm gold clusters. In top views of the groEL complexes, gold densities were observed in the central region; in side views, the densities were seen at the end portions of the cylinders, corresponding to positions within the individual rings. In some cases, two gold densities were observed in the same groEL complex. We conclude that folding intermediates are bound inside central cavities within individual chaperonin rings. In this potentially sequestered location, folding intermediates with a compact conformation can be bound at multiple sites by surrounding monomeric members of the ring; localization of folding within the cavity could also facilitate rebinding of structures that initially fail to incorporate properly into the folding protein.
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- Badcoe I. G., Smith C. J., Wood S., Halsall D. J., Holbrook J. J., Lund P., Clarke A. R. Binding of a chaperonin to the folding intermediates of lactate dehydrogenase. Biochemistry. 1991 Sep 24;30(38):9195–9200. doi: 10.1021/bi00102a010. [DOI] [PubMed] [Google Scholar]
- Barraclough R., Ellis R. J. Protein synthesis in chloroplasts. IX. Assembly of newly-synthesized large subunits into ribulose bisphosphate carboxylase in isolated intact pea chloroplasts. Biochim Biophys Acta. 1980 Jun 27;608(1):19–31. doi: 10.1016/0005-2787(80)90129-x. [DOI] [PubMed] [Google Scholar]
- Bochkareva E. S., Lissin N. M., Girshovich A. S. Transient association of newly synthesized unfolded proteins with the heat-shock GroEL protein. Nature. 1988 Nov 17;336(6196):254–257. doi: 10.1038/336254a0. [DOI] [PubMed] [Google Scholar]
- Buchner J., Schmidt M., Fuchs M., Jaenicke R., Rudolph R., Schmid F. X., Kiefhaber T. GroE facilitates refolding of citrate synthase by suppressing aggregation. Biochemistry. 1991 Feb 12;30(6):1586–1591. doi: 10.1021/bi00220a020. [DOI] [PubMed] [Google Scholar]
- Chandrasekhar G. N., Tilly K., Woolford C., Hendrix R., Georgopoulos C. Purification and properties of the groES morphogenetic protein of Escherichia coli. J Biol Chem. 1986 Sep 15;261(26):12414–12419. [PubMed] [Google Scholar]
- Cheng M. Y., Hartl F. U., Martin J., Pollock R. A., Kalousek F., Neupert W., Hallberg E. M., Hallberg R. L., Horwich A. L. Mitochondrial heat-shock protein hsp60 is essential for assembly of proteins imported into yeast mitochondria. Nature. 1989 Feb 16;337(6208):620–625. doi: 10.1038/337620a0. [DOI] [PubMed] [Google Scholar]
- Creighton T. E. Molecular chaperones. Unfolding protein folding. Nature. 1991 Jul 4;352(6330):17–18. doi: 10.1038/352017a0. [DOI] [PubMed] [Google Scholar]
- Ellis J. Protein folding. Cytosolic chaperonin confirmed. Nature. 1992 Jul 16;358(6383):191–191. doi: 10.1038/358191a0. [DOI] [PubMed] [Google Scholar]
- Ellis R. J., van der Vies S. M. Molecular chaperones. Annu Rev Biochem. 1991;60:321–347. doi: 10.1146/annurev.bi.60.070191.001541. [DOI] [PubMed] [Google Scholar]
- Flynn G. C., Pohl J., Flocco M. T., Rothman J. E. Peptide-binding specificity of the molecular chaperone BiP. Nature. 1991 Oct 24;353(6346):726–730. doi: 10.1038/353726a0. [DOI] [PubMed] [Google Scholar]
- Georgopoulos C. P., Hendrix R. W., Casjens S. R., Kaiser A. D. Host participation in bacteriophage lambda head assembly. J Mol Biol. 1973 May 5;76(1):45–60. doi: 10.1016/0022-2836(73)90080-6. [DOI] [PubMed] [Google Scholar]
- Georgopoulos C. P., Hendrix R. W., Casjens S. R., Kaiser A. D. Host participation in bacteriophage lambda head assembly. J Mol Biol. 1973 May 5;76(1):45–60. doi: 10.1016/0022-2836(73)90080-6. [DOI] [PubMed] [Google Scholar]
- Gething M. J., Sambrook J. Protein folding in the cell. Nature. 1992 Jan 2;355(6355):33–45. doi: 10.1038/355033a0. [DOI] [PubMed] [Google Scholar]
- Goloubinoff P., Christeller J. T., Gatenby A. A., Lorimer G. H. Reconstitution of active dimeric ribulose bisphosphate carboxylase from an unfoleded state depends on two chaperonin proteins and Mg-ATP. Nature. 1989 Dec 21;342(6252):884–889. doi: 10.1038/342884a0. [DOI] [PubMed] [Google Scholar]
- Hainfeld J. F., Furuya F. R. A 1.4-nm gold cluster covalently attached to antibodies improves immunolabeling. J Histochem Cytochem. 1992 Feb;40(2):177–184. doi: 10.1177/40.2.1552162. [DOI] [PubMed] [Google Scholar]
- Hartl F. U., Martin J., Neupert W. Protein folding in the cell: the role of molecular chaperones Hsp70 and Hsp60. Annu Rev Biophys Biomol Struct. 1992;21:293–322. doi: 10.1146/annurev.bb.21.060192.001453. [DOI] [PubMed] [Google Scholar]
- Hendrix R. W. Purification and properties of groE, a host protein involved in bacteriophage assembly. J Mol Biol. 1979 Apr 15;129(3):375–392. doi: 10.1016/0022-2836(79)90502-3. [DOI] [PubMed] [Google Scholar]
- Hohn T., Hohn B., Engel A., Wurtz M., Smith P. R. Isolation and characterization of the host protein groE involved in bacteriophage lambda assembly. J Mol Biol. 1979 Apr 15;129(3):359–373. doi: 10.1016/0022-2836(79)90501-1. [DOI] [PubMed] [Google Scholar]
- Hutchinson E. G., Tichelaar W., Hofhaus G., Weiss H., Leonard K. R. Identification and electron microscopic analysis of a chaperonin oligomer from Neurospora crassa mitochondria. EMBO J. 1989 May;8(5):1485–1490. doi: 10.1002/j.1460-2075.1989.tb03532.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Höll-Neugebauer B., Rudolph R., Schmidt M., Buchner J. Reconstitution of a heat shock effect in vitro: influence of GroE on the thermal aggregation of alpha-glucosidase from yeast. Biochemistry. 1991 Dec 17;30(50):11609–11614. doi: 10.1021/bi00114a001. [DOI] [PubMed] [Google Scholar]
- Ishii N., Taguchi H., Sumi M., Yoshida M. Structure of holo-chaperonin studied with electron microscopy. Oligomeric cpn10 on top of two layers of cpn60 rings with two stripes each. FEBS Lett. 1992 Mar 9;299(2):169–174. doi: 10.1016/0014-5793(92)80240-h. [DOI] [PubMed] [Google Scholar]
- Kaufman B. T. Methotrexate-agarose in the purification of dihydrofolate reductase. Methods Enzymol. 1974;34:272–281. doi: 10.1016/s0076-6879(74)34025-6. [DOI] [PubMed] [Google Scholar]
- Kumar A. A., Blankenship D. T., Kaufman B. T., Freisheim J. H. Primary structure of chicken liver dihydrofolate reductase. Biochemistry. 1980 Feb 19;19(4):667–678. doi: 10.1021/bi00545a010. [DOI] [PubMed] [Google Scholar]
- Laminet A. A., Ziegelhoffer T., Georgopoulos C., Plückthun A. The Escherichia coli heat shock proteins GroEL and GroES modulate the folding of the beta-lactamase precursor. EMBO J. 1990 Jul;9(7):2315–2319. doi: 10.1002/j.1460-2075.1990.tb07403.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Landry S. J., Gierasch L. M. The chaperonin GroEL binds a polypeptide in an alpha-helical conformation. Biochemistry. 1991 Jul 30;30(30):7359–7362. doi: 10.1021/bi00244a001. [DOI] [PubMed] [Google Scholar]
- Langer T., Lu C., Echols H., Flanagan J., Hayer M. K., Hartl F. U. Successive action of DnaK, DnaJ and GroEL along the pathway of chaperone-mediated protein folding. Nature. 1992 Apr 23;356(6371):683–689. doi: 10.1038/356683a0. [DOI] [PubMed] [Google Scholar]
- Langer T., Pfeifer G., Martin J., Baumeister W., Hartl F. U. Chaperonin-mediated protein folding: GroES binds to one end of the GroEL cylinder, which accommodates the protein substrate within its central cavity. EMBO J. 1992 Dec;11(13):4757–4765. doi: 10.1002/j.1460-2075.1992.tb05581.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Martin J., Langer T., Boteva R., Schramel A., Horwich A. L., Hartl F. U. Chaperonin-mediated protein folding at the surface of groEL through a 'molten globule'-like intermediate. Nature. 1991 Jul 4;352(6330):36–42. doi: 10.1038/352036a0. [DOI] [PubMed] [Google Scholar]
- Matthews B. W. Solvent content of protein crystals. J Mol Biol. 1968 Apr 28;33(2):491–497. doi: 10.1016/0022-2836(68)90205-2. [DOI] [PubMed] [Google Scholar]
- McMullin T. W., Hallberg R. L. A highly evolutionarily conserved mitochondrial protein is structurally related to the protein encoded by the Escherichia coli groEL gene. Mol Cell Biol. 1988 Jan;8(1):371–380. doi: 10.1128/mcb.8.1.371. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mendoza J. A., Rogers E., Lorimer G. H., Horowitz P. M. Chaperonins facilitate the in vitro folding of monomeric mitochondrial rhodanese. J Biol Chem. 1991 Jul 15;266(20):13044–13049. [PubMed] [Google Scholar]
- Ostermann J., Horwich A. L., Neupert W., Hartl F. U. Protein folding in mitochondria requires complex formation with hsp60 and ATP hydrolysis. Nature. 1989 Sep 14;341(6238):125–130. doi: 10.1038/341125a0. [DOI] [PubMed] [Google Scholar]
- Saibil H., Dong Z., Wood S., auf der Mauer A. Binding of chaperonins. Nature. 1991 Sep 5;353(6339):25–26. doi: 10.1038/353025b0. [DOI] [PubMed] [Google Scholar]
- Trent J. D., Nimmesgern E., Wall J. S., Hartl F. U., Horwich A. L. A molecular chaperone from a thermophilic archaebacterium is related to the eukaryotic protein t-complex polypeptide-1. Nature. 1991 Dec 12;354(6353):490–493. doi: 10.1038/354490a0. [DOI] [PubMed] [Google Scholar]
- Viitanen P. V., Lorimer G. H., Seetharam R., Gupta R. S., Oppenheim J., Thomas J. O., Cowan N. J. Mammalian mitochondrial chaperonin 60 functions as a single toroidal ring. J Biol Chem. 1992 Jan 15;267(2):695–698. [PubMed] [Google Scholar]
- Wall J. S., Hainfeld J. F. Mass mapping with the scanning transmission electron microscope. Annu Rev Biophys Biophys Chem. 1986;15:355–376. doi: 10.1146/annurev.bb.15.060186.002035. [DOI] [PubMed] [Google Scholar]
- Zeilstra-Ryalls J., Fayet O., Georgopoulos C. The universally conserved GroE (Hsp60) chaperonins. Annu Rev Microbiol. 1991;45:301–325. doi: 10.1146/annurev.mi.45.100191.001505. [DOI] [PubMed] [Google Scholar]
- Zhi W., Landry S. J., Gierasch L. M., Srere P. A. Renaturation of citrate synthase: influence of denaturant and folding assistants. Protein Sci. 1992 Apr;1(4):522–529. doi: 10.1002/pro.5560010407. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zwickl P., Pfeifer G., Lottspeich F., Kopp F., Dahlmann B., Baumeister W. Electron microscopy and image analysis reveal common principles of organization in two large protein complexes: groEL-type proteins and proteasomes. J Struct Biol. 1990 May;103(3):197–203. doi: 10.1016/1047-8477(90)90037-d. [DOI] [PubMed] [Google Scholar]
- van der Vies S. M., Viitanen P. V., Gatenby A. A., Lorimer G. H., Jaenicke R. Conformational states of ribulosebisphosphate carboxylase and their interaction with chaperonin 60. Biochemistry. 1992 Apr 14;31(14):3635–3644. doi: 10.1021/bi00129a012. [DOI] [PubMed] [Google Scholar]