The Hsc66-Hsc20 chaperone system in Escherichia coli: Chaperone activity and interactions with the DnaK-DnaJ-GrpE system

Hsc66, a stress-70 protein, and Hsc20, a J-type accessory protein, comprise a newly described Hsp70-type chaperone system in addition to DnaK-DnaJ-Grp E in Escherichia coil. Because endogenous substrates for the Hsc66-Hsc20 sy stem have not yet been identified, we investigated chaperone-like activitie s of Hsc66 and Hsc20 by their ability to suppress aggregation of denatured model substrate proteins, such as rhodanese, citrate synthase, and lucifera se. Hsc66 suppressed aggregation of rhodanese and citrate synthase, and ATP caused effects consistent with complex destabilization typical of other Hs p70-type chaperones. Differences in the activities of Hsc66 and DnaK, howev er, suggest that these chaperones have dissimilar substrate specificity pro files. Hsc20, unlike DnaJ, did not exhibit intrinsic chaperone activity and appears to function solely as a regulatory cochaperone protein for Hsc66. Possible interactions between the Hsc66-Hsc20 and DnaK-DnaJ-GrpE chaperone systems were also investigated by measuring the effects of cochaperone prot eins on Hsp70 ATPase activities. The nucleotide exchange factor GrpE did no t stimulate the ATPase activity of Hsc66 and thus appears to function speci fically with DnaK. Cross-stimulation by the cochaperones Hsc20 and DnaJ was observed, but the requirement for supraphysiological concentrations makes it unlikely that these interactions occur significantly in vivo. Together t hese results suggest that Hsc66-Hsc20 and DnaK-DnaJ-GrpE comprise separate molecular chaperone systems with distinct, nonoverlapping cellular function s.