IDENTIFICATION OF A CHAPERONIN BINDING-SITE IN A CHLOROPLAST PRECURSOR PROTEIN

Although chaperonin-assisted protein folding has been studied in vitro by a number of investigators, the feature(s) of the unfolded polypeptide that are recognized and bound by chaperonins is not known. We have addressed this question using the precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase (pS) as a substrate for GroEL. The protein was expressed in Escherichia coli as a C-terminal fusion to protein A. Protein A-pS and any associated cellular proteins were then purified by affinity chromatography. GroEL could be eluted from the fusion protein by incubation with ATP and either GroES or casein, consistent with results of in vitro folding assays. At least half of the transit sequence of pS is required to maintain this high stoichiometry of chaperonin binding. Using deletion mutagenesis from the C terminus of pS, we defined the smallest truncation of pS, PAxpS90T, that binds GroEL with high avidity (dissociation constant = 53 nM). A series of site-specific mutations targeting the C-terminal 15-20 amino acids of PAxpS90T was constructed and analyzed for the ability to bind GroEL. Our results show that complex formation between GroEL and pS is not dependent upon any single feature such as overall hydrophobicity, a net positive charge, or secondary structure but may be dependent upon a combination of these features.