Genetic analysis of the bacteriophage T4-encoded cochaperonin Gp31.
Previous genetic and biochemical analyses have established that the bacteriophage T4-encoded Gp31 is a cochaperonin that interacts with Escherichia coli's GroEL to ensure the timely and accurate folding of Gp23, the bacteriophage-encoded major capsid protein. The heptameric Gp31 cochaperonin, like the E. coli GroES cochaperonin, interacts with GroEL primarily through its unstructured mobile loop segment. Upon binding to GroEL, the mobile loop adopts a structured, beta-hairpin turn. In this article, we present extensive genetic data that strongly substantiate and extend these biochemical studies. These studies begin with the isolation of mutations in gene 31 based on the ability to plaque on groEL44 mutant bacteria, whose mutant product interacts weakly with Gp31. Our genetic system is unique because it also allows for the direct selection of revertants of such gene 31 mutations, based on their ability to plaque on groEL515 mutant bacteria. Interestingly, all of these revertants are pseudorevertants because the original 31 mutation is maintained. In addition, we show that the classical tsA70 mutation in gene 31 changes a conserved hydrophobic residue in the mobile loop to a hydrophilic one. Pseudorevertants of tsA70, which enable growth at the restrictive temperatures, acquire the same mutation previously shown to allow plaque formation on groEL44 mutant bacteria. Our genetic analyses highlight the crucial importance of all three highly conserved hydrophobic residues of the mobile loop of Gp31 in the productive interaction with GroEL.
Richardson
A
A
Université de Genève, Département de Biochimie Médicale, Centre Médical Universitaire, CH-1211 Geneva 4, Switzerland. alexandra.richardson@medecine.unige.ch
Georgopoulos
C
C
D000595:Amino Acid Sequence; D017122:Bacteriophage T4 / Q000235:genetics*; D002213:Capsid / Q000096:biosynthesis / Q000737:chemistry; D036022:Capsid Proteins; D018834:Chaperonin 60 / Q000235:genetics / Q000502:physiology; D018833:Chaperonins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D017510:Protein Folding; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology
D036022:Capsid Proteins; D018834:Chaperonin 60; D014764:Viral Proteins; C063473:gene 31 protein, Enterobacteria phage T4; C064517:gp23 protein, Bacteriophage T4; D018833:Chaperonins