GroE-mediated folding of bacterial luciferases in vivo
1993
Escher, A. (Alberta Univ., Edmonton (USA). Dept. of Plant Science. Plant Molecular Genetics Labs.) | Szalay, A.A.
This study indicates that GroE chaperonins mediate de novo protein folding of heterodimeric and monomeric luciferases under heat shock or sub-heat shock conditions in vivo. The effects of additional groESL and groEL genes on the bioluminescence of Escherichia coli cells expressing different bacterial luciferase genes at various temperatures were directly studied in cells growing in liquid culture. Data indicate that at 42 degrees C GroESL chaperonins are required for the folding of the beta subunit polypeptide of the heterodimeric alphabeta luciferase from the mesophilic bacterium Vibrio harveyi MAV (B392). In contrast, the small number of amino acid substitutions present in the luciferase beta subunit polypeptide from the thermotolerant V. harveyi CTP5 suppresses this requirement for GroE chaperonins, and greatly reduced interaction between the beta subunit polypeptide and GroEL chaperonin. In addition, GroESL are required for the de novo folding at 37 degrees C of a MAV alphabeta luciferase fusion polypeptide that is functional as a monomer. No such requirement for luciferase activity is observed at the temperature with a fusion of the CTP5 alpha and beta subunit polypeptides, although GroE chaperonins can still mediate folding of the CTP5 fusion luciferase. Bacterial luciferases provide a unique system for direct observation of the effects of GroE chaperonins on protein folding and enyzme assembly in living cells. Furthermore, they offer a sensitive and simple assay system for the identification of polypeptide domains required for GroEL protein binding
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