Biochemical characterization of ClpB3, a chloroplastic disaggregase from Arabidopsis thaliana

Abstract

Disaggregases from the Hsp100/Clp family are a type of molecular chaperones involved in disassembling protein aggregates. Plant cells are uniquely endowed with ClpB proteins in the cytosol, mitochondria and chloroplasts. Chloroplastic ClpB proteins have been implicated in key processes like the unfolded protein response; however, they have not been studied in detail. In this study, we explored the biochemical properties of a chloroplastic ClpB disaggregase, in particular, ClpB3 from A. thaliana. ClpB3 was produced recombinantly in Escherichia coli and afnity-purifed to near homogeneity. ClpB3 forms a hexameric complex in the presence of MgATP and displays intrinsic ATPase activity. We demonstrate that ClpB3 has ATPase activity in a wide range of pH and temperature values and is particularly resistant to heat. ClpB3 specifcally targets unstructured polypeptides and mediates the reactivation of heat-denatured model substrates without the aid of the Hsp70 system. Overall, this work represents the frst in-depth biochemical description of a ClpB protein from plants and strongly supports its role as the putative disaggregase chaperone in chloroplasts.