Substrate mutations that bypass a specific Cpn10 chaperonin requirement for protein folding.

The bacteriophage T4 GroES homologue, gp31, in conjunction with the Escherichia coli chaperonin GroEL, is both necessary and sufficient to fold the T4 major capsid protein, gp23, to a state competent for capsid assembly as shown by in vivo expression studies. GroES is unable to function in this role as a productive co-chaperonin. The sequencing and characterization of mutations within gp23 that confer GroEL and gp31 chaperonin-independent folding of the mutant protein suggest that the chaperonin requirements are due to specific sequence determinants or structures in critical regions of gp23 that behave in an additive fashion to confer a chaperonin bypass phenotype. Conservative amino acid substitutions in these critical regions enable gp23 to fold in a GroEL-gp31 chaperonin-independent mode, albeit less efficiently than wild type, both in vivo and in vitro. Although the presence of functional GroEL-gp31 enhances folding of the mutated gp23 in vivo, GroEL-GroES has no such effect. Site-directed mutagenesis experiments suggest that a translational pausing mechanism is not responsible for the bypass mutant phenotype. Polyhead reassembly experiments are also consistent with direct, post-translational effects of the bypass mutations on polypeptide folding. Given our finding that gp31 is not required for the binding of the major capsid protein to GroEL and that active GroES is incapable of folding the gp23 polypeptide chain to native conformation, our results suggest co-chaperonin specificity in the folding of certain substrates.