Photosynthesis is performed by large complexes, composed of subunits encoded by the nuclear and chloroplast genomes. Assembly is assisted by general and target-specific chaperones, but their mode of action is yet unclear. We formerly showed that ZnJ2 is an algal chaperone resembling BSD2 from land plants. In algae, it co-migrates with the rbcL transcript on chloroplast polysomes, suggesting it contributes to the de-novo synthesis of RbcL (Doron et al., 2014). ZnJ2 contains four CXXCXGXG motifs, comprising a canonical domain typical also of DnaJ-type I (DNAJA). It contributes to the binding of protein substrates to DnaK and promotes an independent oxidoreductase activity (Mattoo et al., 2014). To examine whether ZnJ2 has oxidoreductase activity, we used the RNaseA assay, which measures the oxidation-dependent reactivation of reduced-denatured RNaseA. Although ZnJ2 assisted the native refolding of reduced-denatured RNaseA, its activity was restricted to an oxidizing environment. Thus, ZnJ2 did not carry the exclusive responsibility for the formation of disulfide bridges, but contributed to the stabilization of its target polypeptides, until they reached their native state. A ZnJ2 cysteine deficient mutant maintained a similar holding chaperone activity as the wild-type and did not induce the formation of disulfide bonds. ZnJ2 is devoid of a J-domain. It thus does not belong to the J-domain co-chaperones that target protein substrates to DnaK. As expected, in vitro, its aggregation-prevention activity was not synergic to the ATP-fueled action of DnaK/DnaJ/GrpE in assisting the native refolding of denatured malate dehydrogenase, nor did it show an independent refolding activity. A phylogenetic analysis showed that ZnJ2 and BSD2 from land plants, are two different proteins belonging to a larger group containing a cysteine-rich domain, that also includes the DNAJAs. Members of this family are apparently involved in specific assembly of photosynthetic complexes in the chloroplast.
Keywords: DnaJ-like chaperone; RNaseA assay; ZnJ2; chloroplast chaperones; redox.