Chloroplast Cpn20 forms a tetrameric structure in Arabidopsis thaliana.
Chloroplast chaperonin 20 (Cpn20) in higher plants is a functional homologue of the Escherichia coli GroES, which is a critical regulator of chaperonin-mediated protein folding. The cDNA for a Cpn20 homologue of Arabidopsis thaliana was isolated. It was 958 bp long, encoding a protein of 253 amino acids. The protein was composed of an N-terminal chloroplast transit peptide, and the predicted mature region comprised two distinct GroES domains that showed 42% amino acid identity to each other. The isolated cDNA was constitutively expressed in transgenic tobacco. Immunogold labelling showed that Cpn20 is accumulated in chloroplasts of transgenic tobacco. A Northern blot analysis revealed that mRNA for the chloroplast Cpn20 is abundant in leaves and is increased by heat treatment. To examine the oligomeric structure of Cpn20, a histidine-tagged construct lacking the transit peptide was expressed in E. coli and purified by affinity chromatography. Gel-filtration and cross-linking analyses showed that the expressed products formed a tetramer. The expressed products could substitute for GroES to assist the refolding of citrate synthase under non-permissive conditions. The analysis on the subunit stoichiometry of the GroEL-Cpn20 complex also revealed that the functional complex is composed of a GroEL tetradecamer and a Cpn20 tetramer.
Koumoto
Y
Y
Department of Cell Biology, National Institute for Basic Biology, Okazaki, Japan.
Shimada
T
T
Kondo
M
M
Takao
T
T
Shimonishi
Y
Y
Hara-Nishimura
I
I
Nishimura
M
M
D017360:Arabidopsis / Q000378:metabolism*; D029681:Arabidopsis Proteins; D001483:Base Sequence; D001704:Biopolymers; D015152:Blotting, Northern; D015139:Blotting, Southern; D018833:Chaperonins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002736:Chloroplasts / Q000378:metabolism*; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D056384:Group I Chaperonins; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation
D029681:Arabidopsis Proteins; D001704:Biopolymers; C478767:Cpn20 protein, Arabidopsis; D017931:DNA Primers; D018076:DNA, Complementary; D018833:Chaperonins; D056384:Group I Chaperonins