ATP‐dependent protein refolding activity in reticulocyte lysate

Abstract
The protein folding capacity of rabbit reticulocyte cytosol was analyzed using the renaturation of firefly luciferase as a sensitive assay. In the absence of ATP, the aggregation of denatured luciferase diluted into reticulocyte lysate was prevented. Chaperone-stabilized luciferase was detected in high molecular weight complexes overlapping the distributions of Hsc70, Hsp90 and the chaperonin TRiC on gel filtration columns. The readdition of unfractionated cytosol and Mg-ATP was required for the efficient folding of these forms of luciferase to the active enzyme. We conclude that protein folding in the eukaryotic cytosol depends on the functional cooperation of different chaperone activities and cofactors in a complex, ATP-dependent process.