Efficient production of native actin upon translation in a bacterial lysate supplemented with the eukaryotic chaperonin TRiC
-
Markus J. Stemp
Abstract
Recombinant expression of actin in bacteria results in non-native species that aggregate into inclusion bodies. Actin is a folding substrate of TRiC, the chaperonin of the eukaryotic cytosol. By employing bacterial in vitro translation lysates supplemented with purified chaperones, we have found that TRiC is the only eukaryotic chaperone necessary for correct folding of newly translated actin. The actin thus produced binds deoxyribonuclease I and polymerizes into filaments, hallmarks of its native state. In contrast to its rapid folding in the eukaryotic cytosol, actin translated in TRiC-supplemented bacterial lysate folds with slower kinetics, resembling the kinetics upon refolding from denaturant. Lysate supplementation with the bacterial chaperonin GroEL/ES or the DnaK/DnaJ/GrpE chaperones leads to prevention of actin aggregation, yet fails to support its correct folding. This combination of in vitro bacterial translation and TRiC-assisted folding allows a detailed analysis of the mechanisms necessary for efficient actin folding in vivo. In addition, it provides a robust alternative for the production of substantial amounts of eukaryotic proteins that otherwise misfold or lead to cellular toxicity upon expression in heterologous hosts.
References
Agashe, V.R., Guha, S., Chang, H.C., Genevaux, P., Hayer-Hartl, M., Stemp, M., Georgopoulos, C., Hartl, F.U., and Barral, J.M. (2004). Function of trigger factor and DnaK in multidomain protein folding: increase in yield at the expense of folding speed. Cell117, 199–209.10.1016/S0092-8674(04)00299-5Suche in Google Scholar PubMed
Anfinsen, C.B. (1973). Principles that govern the folding of protein chains. Science181, 223–230.10.1126/science.181.4096.223Suche in Google Scholar PubMed
Baneyx, F. and Mujacic, M. (2004). Recombinant protein folding and misfolding in Escherichia coli. Nat. Biotechnol.22, 1399–1408.10.1038/nbt1029Suche in Google Scholar PubMed
Blikstad, I., Markey, F., Carlsson, L., Persson, T., and Lindberg, U. (1978). Selective assay of monomeric and filamentous actin in cell extracts, using inhibition of deoxyribonuclease I. Cell15, 935–943.10.1016/0092-8674(78)90277-5Suche in Google Scholar PubMed
Cooper, J.A. (1987). Effects of cytochalasin and phalloidin on actin. J. Cell Biol.105, 1473–1478.10.1083/jcb.105.4.1473Suche in Google Scholar PubMed PubMed Central
Ferreyra, R.G. and Frydman, J. (2000). Purification of the cytosolic chaperonin TRiC from bovine testis. Methods Mol. Biol.140, 153–160.10.1385/1-59259-061-6:153Suche in Google Scholar PubMed
Frydman, J. and Hartl, F.U. (1996). Principles of chaperone-assisted protein folding: differences between in vitro and in vivo mechanisms. Science272, 1497–1502.10.1126/science.272.5267.1497Suche in Google Scholar PubMed
Gao, Y., Thomas, J.O., Chow, R.L., Lee, G.H., and Cowan, N.J. (1992). A cytoplasmic chaperonin that catalyzes β-actin folding. Cell69, 1043–1050.10.1016/0092-8674(92)90622-JSuche in Google Scholar PubMed
Hartl, F.U. and Hayer-Hartl, M. (2002). Molecular chaperones in the cytosol: from nascent chain to folded protein. Science295, 1852–1858.10.1126/science.1068408Suche in Google Scholar PubMed
Hesterkamp, T., Deuerling, E., and Bukau, B. (1997). The amino-terminal 118 amino acids of Escherichia coli trigger factor constitute a domain that is necessary and sufficient for binding to ribosomes. J. Biol. Chem.272, 21865–21871.10.1074/jbc.272.35.21865Suche in Google Scholar PubMed
Hitchcock, S.E., Carisson, L., and Lindberg, U. (1976). Depolymerization of F-actin by deoxyribonuclease I. Cell7, 531–542.10.1016/0092-8674(76)90203-8Suche in Google Scholar PubMed
Llorca, O., Martin-Benito, J., Ritco-Vonsovici, M., Grantham, J., Hynes, G.M., Willison, K.R., Carrascosa, J.L., and Valpuesta, J.M. (2000). Eukaryotic chaperonin CCT stabilizes actin and tubulin folding intermediates in open quasi-native conformations. EMBO J.19, 5971–5979.10.1093/emboj/19.22.5971Suche in Google Scholar PubMed PubMed Central
McCallum, C.D., Do, H., Johnson, A.E., and Frydman, J. (2000). The interaction of the chaperonin tailless complex polypeptide 1 (TCP1) ring complex (TRiC) with ribosome-bound nascent chains examined using photo-cross-linking. J. Cell Biol.149, 591–602.10.1083/jcb.149.3.591Suche in Google Scholar PubMed PubMed Central
Pardee, J.D. and Spudich, J.A. (1982). Purification of muscle actin. Methods Enzymol.85B, 164–181.10.1016/0076-6879(82)85020-9Suche in Google Scholar PubMed
Schägger, H. and von Jagow, G. (1991). Blue native electrophoresis for isolation of membrane protein complexes inenzymatically active form. Anal. Biochem.199, 223–231.10.1016/0003-2697(91)90094-ASuche in Google Scholar
Siegers, K., Waldmann, T., Leroux, M.R., Grein, K., Shevchenko, A., Schiebel, E., and Hartl, F.U. (1999). Compartmentation of protein folding in vivo: sequestration of non-native polypeptide by the chaperonin-GimC system. EMBO J.18, 75–84.10.1093/emboj/18.1.75Suche in Google Scholar PubMed PubMed Central
Siegers, K., Bolter, B., Schwarz, J.P., Bottcher, U.M., Guha, S., and Hartl, F.U. (2003). TRiC/CCT cooperates with different upstream chaperones in the folding of distinct protein classes. EMBO J.22, 5230–5240.10.1093/emboj/cdg483Suche in Google Scholar PubMed PubMed Central
Steinmetz, M.O., Stoffler, D., Muller, S.A., Jahn, W., Wolpensinger, B., Goldie, K.N., Engel, A., Faulstich, H., and Aebi, U. (1998). Evaluating atomic models of F-actin with an undecagold-tagged phalloidin derivative. J. Mol. Biol.276, 1–6.10.1006/jmbi.1997.1529Suche in Google Scholar PubMed
Szabo, A., Langer, T., Schroder, H., Flanagan, J., Bukau, B., and Hartl, F.U. (1994). The ATP hydrolysis-dependent reaction cycle of the Escherichia coli Hsp70 system DnaK, DnaJ, and GrpE. Proc. Natl. Acad. Sci. USA91, 10345–10349.10.1073/pnas.91.22.10345Suche in Google Scholar PubMed PubMed Central
Tian, G., Vainberg, I.E., Tap, W.D., Lewis, S.A., and Cowan, N.J. (1995). Specificity in chaperonin-mediated protein folding. Nature375, 250–253.10.1038/375250a0Suche in Google Scholar PubMed
©2005 by Walter de Gruyter Berlin New York
Artikel in diesem Heft
- Highlight: Molecular Machines
- Splicing of a rare class of introns by the U12-dependent spliceosome
- Proteasome-associated proteins: regulation of a proteolytic machine
- Novel insights into the mechanism of chaperone-assisted protein disaggregation
- Dynamic chromatin: concerted nucleosome remodelling and acetylation
- Efficient production of native actin upon translation in a bacterial lysate supplemented with the eukaryotic chaperonin TRiC
- The NC1 dimer of human placental basement membrane collagen IV: does a covalent crosslink exist?
- Intact growth factors are conserved in the extracellular matrix of ancient human bone and teeth: a storehouse for the study of human evolution in health and disease
- Differential accumulation of plastid preprotein translocon components during spruce (Picea abies L. Karst.) needle development
- Hypothyroidism induces expression of the peptide transporter PEPT2
- The precursor of secreted aspartic proteinase Sapp1p from Candida parapsilosis can be activated both autocatalytically and by a membrane-bound processing proteinase
- Effects of elastase and cathepsin G on the levels of membrane and soluble TNFα
- Isolation and structural characterisation of a novel 13-amino acid insulin-releasing peptide from the skin secretion of Agalychnis calcarifer
Artikel in diesem Heft
- Highlight: Molecular Machines
- Splicing of a rare class of introns by the U12-dependent spliceosome
- Proteasome-associated proteins: regulation of a proteolytic machine
- Novel insights into the mechanism of chaperone-assisted protein disaggregation
- Dynamic chromatin: concerted nucleosome remodelling and acetylation
- Efficient production of native actin upon translation in a bacterial lysate supplemented with the eukaryotic chaperonin TRiC
- The NC1 dimer of human placental basement membrane collagen IV: does a covalent crosslink exist?
- Intact growth factors are conserved in the extracellular matrix of ancient human bone and teeth: a storehouse for the study of human evolution in health and disease
- Differential accumulation of plastid preprotein translocon components during spruce (Picea abies L. Karst.) needle development
- Hypothyroidism induces expression of the peptide transporter PEPT2
- The precursor of secreted aspartic proteinase Sapp1p from Candida parapsilosis can be activated both autocatalytically and by a membrane-bound processing proteinase
- Effects of elastase and cathepsin G on the levels of membrane and soluble TNFα
- Isolation and structural characterisation of a novel 13-amino acid insulin-releasing peptide from the skin secretion of Agalychnis calcarifer
