Co-chaperonin specificity in the folding of the bacteriophage T4 major capsid protein

Abstract

Bacteriophage T4 is unique among phages in that its growth only requires the Escherichia coli GroEL (cpn60) protein but not the GroES (cpn10) co-chaperonin. Our findings indicate that bacteriophage T4 synthesizes its own co-chaperonin, gp31, which in conjunction with GroEL, is strictly required for the folding of the T4 major capsid protein, gp23. Overexpression work demonstrates that GroEL and gp31 are both necessary and sufficient for the proper folding and oligomerization of the major capsid protein in vivo. Although gp31 and GroES have no significant sequence homology, either on the primary amino acid level or on the predicted secondary structural level, the two proteins appear to be functional homologues. Cryoelectron microscopy of gp31 purified to 90% indicates gp31 forms oligomeric ring structures that are comparable to those formed by GroES. Studies using expression vectors and genetic analysis indicate that although the GroEL-gp31 complex can fold proteins normally folded by GroEL-GroES, the reverse is not true. Thus, even when GroES is overproduced it is unable to mediate chaperonin-assisted folding of the T4 major capsid protein. In addition, co-immunoprecipitation experiments using antisera directed against GroEL and GroES, suggest that GroES protein is functional during T4 infection and able to bind GroEL protein. These studies demonstrate that gp31 is specifically required to fold the T4 major capsid protein because of a difference in co-chaperonin function and not because GroES protein is inactivated or limiting during the course of T4 infection. In addition, co-immunoprecipitation experiments demonstrate that although gp31 is required for the folding of the T4 major capsid protein, gp31 is not required for the binding of the major capsid protein to GroEL. T4bypass31 are mutant phage that can bypass the strict chaperonin requirement for growth. The presence of both the bypass 31-1 and bypass 31-2 mutations in gp23 (BY23) allows the major capsid protein to fold, although less efficiently, in a GroEL-gp31 chaperonin independent mode. The mutations that confer the bypass phenotype have now been sequenced to two specific regions of the gene encoding the major capsid protein which appear to be critical folding sites of the polypeptide as judged by previous genetic analysis. The bypass31-1 mutation is a single missense mutation near the 3 prime end of gene that results in the conversion of Ala-455 to Val-455. The bypass31-2 site, located in the center of gene 23, consists of three independent missense mutations that convert Gly-292 to Ser-292, Val-306 to Ile-306, and Val-307 to Ile-307. Characterization of these mutations by site-directed mutagenesis and genetic studies show that all three mutations at the bypass31-2 site are required for optimal phage growth in the absence of GroEL and gp31, but that Ile-306 and Ile-307 are essential to maintain the bypass phenotype. In addition, all four bypass mutations behave additively at elevated temperatures. Mutational analysis also suggests that the bypass phenotype is inherent to the gp23 polypeptide and is not due to translational pausing. (Abstract shortened by UMI.)