title: Functional analysis of isolated cpn10 domains and conserved amino acid residues in spinach chloroplast co-chaperonin by site-directed mutagenesis.
abstract: The possibilities of independent function of the two chaperonin 10 (cpn10) domains of the cpn10 homologue from spinach chloroplasts and the role of five conserved amino acid residues in the N-terminal cpn10 unit were investigated. Recombinant single domain proteins and complete chloroplast cpn10 proteins carrying amino acid exchanges of conserved residues in their N-terminal cpn10 domain were expressed in Escherichia coli and partially purified. The function of the recombinant proteins was tested using GroEL as chaperonin 60 (cpn60) partner for in vitro refolding of denatured ribulose-1,5-bisphosphate carboxylase (Rubisco). Interaction with cpn60 was also monitored by the ability to inhibit GroEL ATPase activity. In vitro both isolated cpn10 domains were found to be incapable of co-chaperonin function. All mutants were also severely impaired in cpn10 function. The results are interpreted in terms of an essential role of the exchanged amino acid residues for the interaction between co-chaperonin and cpn60 partner and in terms of a functional coupling of both cpn10 domains. To test the function of mutant chloroplast cpn10 proteins in vivo the cpn10 deficiency of E. coli strain CG712 resulting in an inability to assemble lambda-phage was exploited in a complementation assay. Transformation with plasmids directing the expression of mutant chloroplas cpn10 proteins in two cases restored lambda-phage assembly in this bacterial strain to the same extent as did transformation with a plasmid encoding wild-type cpn10 protein. In contrast a plasmid encoded third mutant and truncated forms of chloroplast cpn10 showed significantly reduced complementation efficiencies.
authors.affiliation: Botanisches Institut, Christian-Albrechts-Universität, Kiel, Germany.
authors.forename: U
authors.initials: U
authors.lastname: Bertsch
authors.forename: J
authors.initials: J
authors.lastname: Soll
mesh_terms: D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors; D000595:Amino Acid Sequence; D001483:Base Sequence; D018835:Chaperonin 10 / Q000235:genetics* / Q000378:metabolism; D018834:Chaperonin 60 / Q000235:genetics / Q000302:isolation & purification; D002736:Chloroplasts / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D017510:Protein Folding; D012273:Ribulose-Bisphosphate Carboxylase / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D018724:Spinacia oleracea / Q000235:genetics*
chemical_list: D018835:Chaperonin 10; D018834:Chaperonin 60; D018076:DNA, Complementary; D000251:Adenosine Triphosphatases; D012273:Ribulose-Bisphosphate Carboxylase