GroEL and GroES control of substrate flux in the in vivo folding pathway of phage P22 coat protein.
Our present understanding of the action of the chaperonins GroEL/S on protein folding is based primarily on in vitro studies, whereas the folding of proteins in the cellular milieu has not been as thoroughly investigated. We have developed a means of examining in vivo protein folding and assembly that utilizes the coat protein of bacteriophage P22, a naturally occurring substrate of GroEL/S. Here we show that amino acid substitutions in coat protein that cause a temperature-sensitive-folding (tsf) phenotype slowed assembly rates upon increasing the temperature of cell growth. Raising cellular concentrations of GroEL/S increased the rate of assembly of the tsf mutant coat proteins to nearly that of wild-type (WT) coat protein by protecting a thermolabile folding intermediate from aggregation, thereby increasing the concentration of assembly-competent coat protein. The rate of release of the tsf coat proteins from the GroEL/S-coat protein ternary complex was approximately 2-fold slower at non-permissive temperatures when compared with the release of WT coat protein. However, the rate of release of WT or tsf coat proteins at each temperature remained constant regardless of GroEL/S levels. Thus, raising the cellular concentration of GroEL/S increased the amount of assembly-competent tsf coat proteins not by altering the rates of folding but by increasing the probability of GroEL/S-coat protein complex formation.
Nakonechny
W S
WS
Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269-3125, USA.
Teschke
C M
CM
D017100:Bacteriophage P22; D002213:Capsid / Q000235:genetics / Q000378:metabolism*; D018835:Chaperonin 10 / Q000378:metabolism*; D018834:Chaperonin 60 / Q000378:metabolism*; D004926:Escherichia coli / Q000821:virology; D008954:Models, Biological; D009154:Mutation; D010641:Phenotype; D011485:Protein Binding; D017510:Protein Folding; D012486:Salmonella typhimurium / Q000821:virology; D013696:Temperature
D018835:Chaperonin 10; D018834:Chaperonin 60