FUNCTIONAL-ANALYSIS OF ISOLATED CPN10 DOMAINS AND CONSERVED AMINO-ACID-RESIDUES IN SPINACH CHLOROPLAST CO-CHAPERONIN BY SITE-DIRECTED MUTAGENESIS

The possibilities of independent function of the two chaperonin 10 (cp n10) domains of the cpn10 homologue from spinach chloroplasts and the role of five conserved amino acid residues in the N-terminal cpn10 uni t were investigated. Recombinant single domain proteins and complete c hloroplast cpn10 proteins carrying amino acid exchanges of conserved r esidues 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 seve rely impaired in cpn10 function. The results are interpreted in terms of an essential role of the exchanged amino acid residues for the inte raction between co-chaperonin and cpn60 partner and in terms of a func tional coupling of both cpn10 domains. To test the function of mutant chloroplast cpn10 proteins in vivo the cpn10 deficiency of E. coli str ain CG712 resulting in an inability to assemble lambda-phage was explo ited in a complementation assay. Transformation with plasmids directin g the expression of mutant chloroplast cpn10 proteins in two cases res tored lambda-phage assembly in this bacterial strain to the same exten t as did transformation with a plasmid encoding wild-type cpn10 protei n. In contrast a plasmid encoded third mutant and truncated forms of c hloroplast cpn10 showed significantly reduced complementation efficien cies.