In vitro characterization of bacterial and chloroplast HSP70 systems reveals an evolutionary optimization of the co-chaperones for their HSP70 partner

The chloroplast Hsp70 system involved in protein folding in Chlamydomonas reinhardtii consists of HSP70B, the DnaJ homolog CDJ1, and the GrpE-type nucleotide exchange factor CGE1. The finding that HSP70B needs to be co-expressed with the escort protein HEP2 to become functional allowed reconstituting the chloroplast Hsp70 system in vitro and comparing it with the homologous E. coli system. Both systems support luciferase refolding and display ATPase and holdase activities. Steady-state activities are low and strongly stimulated by the co-chaperones, whose concentrations need to be balanced to optimally support luciferase refolding. Although the co-chaperones of either system generally stimulate ATPase and folding assistance activities of the other, luciferase refolding is reduced ~10-fold and <2-fold if either Hsp70 is supplemented with the foreign DnaJ and GrpE protein, respectively, suggesting an evolutionary specialization of the co-chaperones for their Hsp70 partner. Distinct features are that HSP70B’s steady-state ATPase exhibits ~20-fold higher values for Vmax and Km, and that the HSP70B system displays a ~6-fold higher folding assistance on denatured luciferase. While truncating up to 16 N-terminal amino acids of CGE1 does not affect HSP70B’s general ATPase and folding assistance activities in the physiological temperature range, further deletions hampering dimerization of CGE1 via its N-terminal coiled-coil do.
Source: BJ Cell - Category: Biochemistry Authors: Tags: BJ Plant Source Type: research