CONSERVED ATPASE AND LUCIFERASE REFOLDING ACTIVITIES BETWEEN BACTERIAAND YEAST HSP70 CHAPERONES AND MODULATORS

We have reconstituted an ATP-dependent protein folding machinery using purified yeast cytosolic proteins. The S. cerevisiae Hsp70 Ssa1p and the DnaJ homolog Ydj1p refolded denatured firefly luciferase. In E. co li, efficient refolding of luciferase requires the Hsp7O DnaK and two modulators, DnaJ and GrpE, that synergistically stimulate its ATPase a ctivity. Exchanging DnaJ homologs between the S. cerevisiae and E. col i systems revealed that their ability to stimulate Hsp70 ATPase activi ty was conserved. In contrast, GrpE further stimulated only DnaK's ATP ase activity. Efficient refolding of luciferase by Ssa1p and DnaJ, but not by DnaK and Ydj1p, suggests that a compatible Hsp70/DnaJ homolog pair can act as a protein folding machinery.