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cDNA sequence and overexpression of chloroplast chaperonin 21 from Arabidopsis thaliana
Biochimica et Biophysica Acta 1429 (1999) 512^515
Short sequence-paper
cDNA sequence and overexpression of chloroplast chaperonin 21 from Arabidopsis thaliana Toshiya Hirohashi, Kazuaki Nishio, Masato Nakai * Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565, Japan Received 6 October 1998; accepted 24 November 1998
Abstract Higher plant chloroplasts contain a 21-kDa protein, chaperonin 21 (Cpn21), that is a functional homolog of the chaperonin 10 (Cpn10). The chloroplast Cpn21 polypeptide consists of two Cpn10-like domains fused together in tandem. We describe here the cDNA sequence of the Cpn21 (AtCpn21) precursor protein from Arabidopsis thaliana. The deduced amino acid sequence of the AtCpn21 precursor protein, 253 amino acids long, shows 61% identity with the spinach Cpn21 protein. The AtCpn21 precursor protein contains the typical chloroplast transit peptide of 51 amino acids at its aminoterminus and the two Cpn10-like domains which exhibits 46% sequence identity to each other. The predicted mature-sized polypeptide of AtCpn21 was expressed in Escherichia coli as a soluble 21-kDa protein. Gel-filtration and chemical crosslinking analyses showed that the recombinant mature AtCpn21 protein forms a stable homo-oligomer composed of three or four polypeptides. ß 1999 Elsevier Science B.V. All rights reserved. Keywords: Molecular chaperone; Chaperonin; Chloroplast ; Arabidopsis thaliana
The best-studied molecular chaperone, the chaperonin protein, consists of two family members: GroEL or chaperonin 60 (Cpn60) and GroES or chaperonin 10 (Cpn10). Both proteins are essential for full chaperonin function in vivo [1,2]. Biochemical and structural analyses revealed that the Cpn60 forms two 7-fold rotationally symmetric rings each of which contains seven identical Cpn60 subunits [3]. Inside a large central cavity within each ring, the folding of a large variety of newly synthesized proteins is thought to be promoted [3]. Cpn10 also forms a seven-subunit homo-oligomeric ring structure and associates with the Cpn60 oligomer in an
* Corresponding author. Fax: +81 (6) 879-8613; E-mail: [email protected]
ATP-dependent manner during a Cpn60^Cpn10mediated protein folding reaction [4,5]. Higher plant chloroplast chaperonin has been known to exhibit characteristic protein composition [6^10]: (1) two distinct Cpn60 polypeptides, Cpn60-K and Cpn60-L, are present in nearly equal amounts and form a hetero-oligomer (Nishio et al., manuscript in preparation); and (2) chloroplast Cpn10 homolog, termed Cpn21, is a `double' Cpn10 molecule in tandem whose subunit molecular mass is about twice that of the bacterial or mitochondrial Cpn10. Although some biochemical analyses of chloroplast Cpn21 protein have been carried out [10^12], the detailed structure^function relationship of Cpn21 remains to be characterized. Arabidopsis thaliana EST clone, 138B2T7, which seemed to contain the full-length cDNA encoding
0167-4838 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 8 3 8 ( 9 8 ) 0 0 2 6 8 - 4
BBAPRO 30427 29-12-98
T. Hirohashi et al. / Biochimica et Biophysica Acta 1429 (1999) 512^515
513
Fig. 1. cDNA and predicted amino acid sequences containing the Arabidopsis thaliana Cpn21. The nucleotide sequence shown here has been assigned EMBL/GenBank accession number AJ010818.
the chloroplast Cpn21 precursor protein, was provided by Arabidopsis Genetic Stock Center. The complete cDNA and deduced amino acid sequences are shown in Fig. 1 and have been deposited in the EMBL/GenBank Data Libraries with accession number AJ010818. The nucleotide sequence contains an open reading frame of 253 amino acids and 5P- and 3P-non-coding regions of 149- and 141-bp, respectively. The deduced amino acid sequence of Arabidopsis Cpn21 (AtCpn21) precursor has a close identity (61%) to the spinach Cpn21 precursor as shown in Fig. 2. By comparison, of the deduced sequence with the amino-terminal amino acid sequences of mature Cpn21 proteins prepared from pea or spinach chloroplasts, the amino-terminal 51 amino acids of the AtCpn21 seems to be a transit peptide for intracellular localization into chloroplasts. The predicted
mature AtCpn21 moiety is composed of the two Cpn10-like domains which are connected by short linker amino acids. The two Cpn10-like domains show 46% sequence identity to each other whereas they show more close similarities to the corresponding domains of the spinach Cpn21 protein; the amino-terminal halves (Cpn21-N) show 62% identity, and the carboxyl-terminal halves (Cpn21-C) show 75% identity (Table 1). To characterize the chloroplast Cpn21 protein in more detail, obtaining a large amount of puri¢ed protein is a prerequisite. Thus, we cloned the portion of cDNA of AtCpn21 corresponding to the predicted mature polypeptide into an Escherichia coli expression vector, pET21d (Novagen), and an over-expression experiment was carried out in E. coli BL21(DE3). The recombinant AtCpn21 protein was
Fig. 2. Alignment of amino acid sequence of the predicted Arabidopsis thaliana Cpn21 with that of Spinacia oleracea Cpn21 [10]. The sequences of the putative amino-terminal chloroplast transit peptides are underlined. The identical amino acids are indicated with asterisks. Boxes de¢ne short linker sequences between two Cpn10-like domains.
BBAPRO 30427 29-12-98
514
T. Hirohashi et al. / Biochimica et Biophysica Acta 1429 (1999) 512^515
Table 1 Percentage of identical amino acids Cpn21-N (At) Cpn21-C (At) Cpn21-N (So) Cpn21-C (So)
GroES (Ec)
Cpn21-N (At)
Cpn21-C (At)
Cpn21-N (So)
38.5 36.2 36.5 33.0
46.2 62.1 38.7
40.9 75.0
39.8
At, Arabidopsis thaliana; So, Spinacia oleracea; Ec, Escherichia coli.
expressed as a 21-kDa protein at a level of almost 50% of total proteins of the E. coli cells (data not shown). The AtCpn21 was recovered mainly in a soluble fraction and puri¢ed to homogeneity by ion-exchange chromatography with MonoS (Pharmacia) as previously described [11] (data not shown). Upon gel ¢ltration through a Superdex 200 (Pharmacia) column, the puri¢ed AtCpn21 displayed a native molecular mass of 70^80 kDa (data not shown). The estimated molecular mass was in good agreement with that found in the case of the authentic spinach Cpn21 [12]. The oligomeric structure of the AtCpn21 protein was further analyzed by chemical cross-linking experiments. As shown in Fig. 3, incubating the
puri¢ed AtCpn21 with water-insoluble cross-linker DSG yielded four discrete bands observed by SDSPAGE. Water-soluble cross-linker BS3 also gave essentially the same results (data not shown). These results suggest that the recombinant AtCpn21 protein forms a tetrameric structure in the E. coli cells. In conclusion, we presented the cDNA sequence of Cpn21 precursor protein from A. thaliana and demonstrated the production of the mature-size recombinant protein which most likely forms a native tetrameric structure in E. coli. The puri¢ed AtCpn21 oligomer might be useful in the further characterization of the structure^function relationship of the higher plant chloroplast chaperonin. This work was supported in part by a grant from a Grant-in-Aid for Scienti¢c Research from the Ministry of Education, Science and Culture of Japan.
References
Fig. 3. Chemical cross-linking of the recombinant AtCpn21 protein. Puri¢ed Cpn21 protein was cross-linked with the indicated concentration of the water-insoluble cross-linker, DSG. Reactions were terminated by the addition of excess Tris base and analyzed by 10% SDS-PAGE and Coomassie brilliant blue staining.
[1] B. Bukau, A.L. Horwich, The Hsp70 and Hsp60 chaperone machines, Cell 92 (1998) 351^366. [2] F.U. Hartl, Molecular chaperones in cellular protein folding, Nature 381 (1996) 571^580. [3] W.A. Fenton, A.L. Horwich, GroEL-mediated protein folding, Protein Sci. 6 (1997) 743^760. [4] Z. Xu, A.L. Horwich, P.B. Sigler, The crystal structure of the asymmetric GroEL^GroES^(ADP)7 chaperonin complex, Nature 388 (1997) 741^750. [5] J.S. Weissman, C.M. Hohl, O. Kovalenko, Y. Kashi, S. Chen, K. Braig, H.R. Saibil, W.A. Fenton, A.L. Horwich, Characterization of the active intermediate of a GroEL^ GroES-mediated protein folding reaction, Cell 84 (1996) 481^490. [6] J.E. Musgrove, R.A. Johnson, R.J. Ellis, Dissociation of the ribulosebisphosphate-carboxylase large-subunit binding protein into dissimilar subunits, Eur. J. Biochem. 163 (1987) 529^534. [7] R. Martel, L.P. Cloney, L.E. Pelcher, S.M. Hemmingsen, Unique composition of plastid chaperonin-60: K and L poly-
BBAPRO 30427 29-12-98
T. Hirohashi et al. / Biochimica et Biophysica Acta 1429 (1999) 512^515 pepide-encoding genes are highly divergent, Gene 94 (1990) 181^187. [8] P.V. Viitanen, M. Schmidt, J. Buchner, T. Suzuki, E. Vierling, R. Dickson, G.H. Lorimer, A. Gatenby, J. Soll, Functional characterization of the higher plant chloroplast chaperonins, J. Biol. Chem. 270 (1995) 18158^18164. [9] L.P. Cloney, H.B. Wu, S.M. Hemmingsen, Expression of plant chaperonin-60 genes in Escherichia coli, J. Biol. Chem. 267 (1992) 23327^23332. [10] U. Bertsch, J. Soll, R. Seetharam, P.V. Viitanen, Identi¢cation, characterization, and DNA sequence of a functional `double' groES-like chaperonin from chloroplasts of higher plants, Proc. Natl. Acad. Sci. USA 89 (1992) 8696^8700.
515
[11] F. Baneyx, U. Bertsch, C.E. Kalbach, S.M. van der Vies, J. Soll, A.A. Gatenby, Spinach chloroplast cpn21 co-chaperonin possesses two functional domains fused together in a toroidal structure and exhibits nucleotide-dependent binding to plastid chaperonin 60, J. Biol. Chem. 279 (1995) 10695^ 10702. [12] M.T. Ryan, D.J. Naylor, N.J. Hoogenraad, P.B. HÖj, A¤nity puri¢cation, overexpression, and characterization of chaperonin 10 homologues synthesized with and without N-terminal acetylation, J. Biol. Chem. 270 (1995) 22037^ 22043.
BBAPRO 30427 29-12-98
Short sequence-paper
cDNA sequence and overexpression of chloroplast chaperonin 21 from Arabidopsis thaliana Toshiya Hirohashi, Kazuaki Nishio, Masato Nakai * Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565, Japan Received 6 October 1998; accepted 24 November 1998
Abstract Higher plant chloroplasts contain a 21-kDa protein, chaperonin 21 (Cpn21), that is a functional homolog of the chaperonin 10 (Cpn10). The chloroplast Cpn21 polypeptide consists of two Cpn10-like domains fused together in tandem. We describe here the cDNA sequence of the Cpn21 (AtCpn21) precursor protein from Arabidopsis thaliana. The deduced amino acid sequence of the AtCpn21 precursor protein, 253 amino acids long, shows 61% identity with the spinach Cpn21 protein. The AtCpn21 precursor protein contains the typical chloroplast transit peptide of 51 amino acids at its aminoterminus and the two Cpn10-like domains which exhibits 46% sequence identity to each other. The predicted mature-sized polypeptide of AtCpn21 was expressed in Escherichia coli as a soluble 21-kDa protein. Gel-filtration and chemical crosslinking analyses showed that the recombinant mature AtCpn21 protein forms a stable homo-oligomer composed of three or four polypeptides. ß 1999 Elsevier Science B.V. All rights reserved. Keywords: Molecular chaperone; Chaperonin; Chloroplast ; Arabidopsis thaliana
The best-studied molecular chaperone, the chaperonin protein, consists of two family members: GroEL or chaperonin 60 (Cpn60) and GroES or chaperonin 10 (Cpn10). Both proteins are essential for full chaperonin function in vivo [1,2]. Biochemical and structural analyses revealed that the Cpn60 forms two 7-fold rotationally symmetric rings each of which contains seven identical Cpn60 subunits [3]. Inside a large central cavity within each ring, the folding of a large variety of newly synthesized proteins is thought to be promoted [3]. Cpn10 also forms a seven-subunit homo-oligomeric ring structure and associates with the Cpn60 oligomer in an
* Corresponding author. Fax: +81 (6) 879-8613; E-mail: [email protected]
ATP-dependent manner during a Cpn60^Cpn10mediated protein folding reaction [4,5]. Higher plant chloroplast chaperonin has been known to exhibit characteristic protein composition [6^10]: (1) two distinct Cpn60 polypeptides, Cpn60-K and Cpn60-L, are present in nearly equal amounts and form a hetero-oligomer (Nishio et al., manuscript in preparation); and (2) chloroplast Cpn10 homolog, termed Cpn21, is a `double' Cpn10 molecule in tandem whose subunit molecular mass is about twice that of the bacterial or mitochondrial Cpn10. Although some biochemical analyses of chloroplast Cpn21 protein have been carried out [10^12], the detailed structure^function relationship of Cpn21 remains to be characterized. Arabidopsis thaliana EST clone, 138B2T7, which seemed to contain the full-length cDNA encoding
0167-4838 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 8 3 8 ( 9 8 ) 0 0 2 6 8 - 4
BBAPRO 30427 29-12-98
T. Hirohashi et al. / Biochimica et Biophysica Acta 1429 (1999) 512^515
513
Fig. 1. cDNA and predicted amino acid sequences containing the Arabidopsis thaliana Cpn21. The nucleotide sequence shown here has been assigned EMBL/GenBank accession number AJ010818.
the chloroplast Cpn21 precursor protein, was provided by Arabidopsis Genetic Stock Center. The complete cDNA and deduced amino acid sequences are shown in Fig. 1 and have been deposited in the EMBL/GenBank Data Libraries with accession number AJ010818. The nucleotide sequence contains an open reading frame of 253 amino acids and 5P- and 3P-non-coding regions of 149- and 141-bp, respectively. The deduced amino acid sequence of Arabidopsis Cpn21 (AtCpn21) precursor has a close identity (61%) to the spinach Cpn21 precursor as shown in Fig. 2. By comparison, of the deduced sequence with the amino-terminal amino acid sequences of mature Cpn21 proteins prepared from pea or spinach chloroplasts, the amino-terminal 51 amino acids of the AtCpn21 seems to be a transit peptide for intracellular localization into chloroplasts. The predicted
mature AtCpn21 moiety is composed of the two Cpn10-like domains which are connected by short linker amino acids. The two Cpn10-like domains show 46% sequence identity to each other whereas they show more close similarities to the corresponding domains of the spinach Cpn21 protein; the amino-terminal halves (Cpn21-N) show 62% identity, and the carboxyl-terminal halves (Cpn21-C) show 75% identity (Table 1). To characterize the chloroplast Cpn21 protein in more detail, obtaining a large amount of puri¢ed protein is a prerequisite. Thus, we cloned the portion of cDNA of AtCpn21 corresponding to the predicted mature polypeptide into an Escherichia coli expression vector, pET21d (Novagen), and an over-expression experiment was carried out in E. coli BL21(DE3). The recombinant AtCpn21 protein was
Fig. 2. Alignment of amino acid sequence of the predicted Arabidopsis thaliana Cpn21 with that of Spinacia oleracea Cpn21 [10]. The sequences of the putative amino-terminal chloroplast transit peptides are underlined. The identical amino acids are indicated with asterisks. Boxes de¢ne short linker sequences between two Cpn10-like domains.
BBAPRO 30427 29-12-98
514
T. Hirohashi et al. / Biochimica et Biophysica Acta 1429 (1999) 512^515
Table 1 Percentage of identical amino acids Cpn21-N (At) Cpn21-C (At) Cpn21-N (So) Cpn21-C (So)
GroES (Ec)
Cpn21-N (At)
Cpn21-C (At)
Cpn21-N (So)
38.5 36.2 36.5 33.0
46.2 62.1 38.7
40.9 75.0
39.8
At, Arabidopsis thaliana; So, Spinacia oleracea; Ec, Escherichia coli.
expressed as a 21-kDa protein at a level of almost 50% of total proteins of the E. coli cells (data not shown). The AtCpn21 was recovered mainly in a soluble fraction and puri¢ed to homogeneity by ion-exchange chromatography with MonoS (Pharmacia) as previously described [11] (data not shown). Upon gel ¢ltration through a Superdex 200 (Pharmacia) column, the puri¢ed AtCpn21 displayed a native molecular mass of 70^80 kDa (data not shown). The estimated molecular mass was in good agreement with that found in the case of the authentic spinach Cpn21 [12]. The oligomeric structure of the AtCpn21 protein was further analyzed by chemical cross-linking experiments. As shown in Fig. 3, incubating the
puri¢ed AtCpn21 with water-insoluble cross-linker DSG yielded four discrete bands observed by SDSPAGE. Water-soluble cross-linker BS3 also gave essentially the same results (data not shown). These results suggest that the recombinant AtCpn21 protein forms a tetrameric structure in the E. coli cells. In conclusion, we presented the cDNA sequence of Cpn21 precursor protein from A. thaliana and demonstrated the production of the mature-size recombinant protein which most likely forms a native tetrameric structure in E. coli. The puri¢ed AtCpn21 oligomer might be useful in the further characterization of the structure^function relationship of the higher plant chloroplast chaperonin. This work was supported in part by a grant from a Grant-in-Aid for Scienti¢c Research from the Ministry of Education, Science and Culture of Japan.
References
Fig. 3. Chemical cross-linking of the recombinant AtCpn21 protein. Puri¢ed Cpn21 protein was cross-linked with the indicated concentration of the water-insoluble cross-linker, DSG. Reactions were terminated by the addition of excess Tris base and analyzed by 10% SDS-PAGE and Coomassie brilliant blue staining.
[1] B. Bukau, A.L. Horwich, The Hsp70 and Hsp60 chaperone machines, Cell 92 (1998) 351^366. [2] F.U. Hartl, Molecular chaperones in cellular protein folding, Nature 381 (1996) 571^580. [3] W.A. Fenton, A.L. Horwich, GroEL-mediated protein folding, Protein Sci. 6 (1997) 743^760. [4] Z. Xu, A.L. Horwich, P.B. Sigler, The crystal structure of the asymmetric GroEL^GroES^(ADP)7 chaperonin complex, Nature 388 (1997) 741^750. [5] J.S. Weissman, C.M. Hohl, O. Kovalenko, Y. Kashi, S. Chen, K. Braig, H.R. Saibil, W.A. Fenton, A.L. Horwich, Characterization of the active intermediate of a GroEL^ GroES-mediated protein folding reaction, Cell 84 (1996) 481^490. [6] J.E. Musgrove, R.A. Johnson, R.J. Ellis, Dissociation of the ribulosebisphosphate-carboxylase large-subunit binding protein into dissimilar subunits, Eur. J. Biochem. 163 (1987) 529^534. [7] R. Martel, L.P. Cloney, L.E. Pelcher, S.M. Hemmingsen, Unique composition of plastid chaperonin-60: K and L poly-
BBAPRO 30427 29-12-98
T. Hirohashi et al. / Biochimica et Biophysica Acta 1429 (1999) 512^515 pepide-encoding genes are highly divergent, Gene 94 (1990) 181^187. [8] P.V. Viitanen, M. Schmidt, J. Buchner, T. Suzuki, E. Vierling, R. Dickson, G.H. Lorimer, A. Gatenby, J. Soll, Functional characterization of the higher plant chloroplast chaperonins, J. Biol. Chem. 270 (1995) 18158^18164. [9] L.P. Cloney, H.B. Wu, S.M. Hemmingsen, Expression of plant chaperonin-60 genes in Escherichia coli, J. Biol. Chem. 267 (1992) 23327^23332. [10] U. Bertsch, J. Soll, R. Seetharam, P.V. Viitanen, Identi¢cation, characterization, and DNA sequence of a functional `double' groES-like chaperonin from chloroplasts of higher plants, Proc. Natl. Acad. Sci. USA 89 (1992) 8696^8700.
515
[11] F. Baneyx, U. Bertsch, C.E. Kalbach, S.M. van der Vies, J. Soll, A.A. Gatenby, Spinach chloroplast cpn21 co-chaperonin possesses two functional domains fused together in a toroidal structure and exhibits nucleotide-dependent binding to plastid chaperonin 60, J. Biol. Chem. 279 (1995) 10695^ 10702. [12] M.T. Ryan, D.J. Naylor, N.J. Hoogenraad, P.B. HÖj, A¤nity puri¢cation, overexpression, and characterization of chaperonin 10 homologues synthesized with and without N-terminal acetylation, J. Biol. Chem. 270 (1995) 22037^ 22043.
BBAPRO 30427 29-12-98