PT - JOURNAL ARTICLE AU - Richa Amiya AU - Michal Shapira TI - ZnJ6 is a DnaJ-like Chaperone with Oxidizing Activity in the Thylakoid Membrane in <em>Chlamydomonas reinhardtii</em> AID - 10.1101/2020.09.14.296681 DP - 2020 Jan 01 TA - bioRxiv PG - 2020.09.14.296681 4099 - http://biorxiv.org/content/early/2020/09/15/2020.09.14.296681.short 4100 - http://biorxiv.org/content/early/2020/09/15/2020.09.14.296681.full AB - Assembly of photosynthetic complexes is sensitive to changes in light intensities, drought, and pathogens that induce a redox imbalance, and require a variety of substrate-specific chaperones to overcome the stress. Proteins with cysteine (C) residues and disulfide bridges are more responsive to the redox changes. This study reports on a thylakoid membrane-associated DnaJ-like protein, ZnJ6 (ZnJ6.g251716.t1.2) in Chlamydomonas reinhardtii. The protein has four CXXCX(G)X(G) motifs that form a functional zinc-binding domain. Site-directed mutagenesis (Cys to Ser) in all the CXXCX(G)X(G) motifs eliminates its zinc-binding ability. In vitro chaperone assays using recombinant ZnJ6 confirm that it is a chaperone that possesses both holding and oxidative refolding activities. Although mutations (Cys to Ser) do not affect the holding activity of ZnJ6, they impair its ability to promote redox-controlled reactivation of reduced and denatured RNaseA, a common substrate protein. The presence of an intact zinc-binding domain is also required for protein stability at elevated temperatures, as suggested by a single spectrum melting curve. Pull-down assays with recombinant ZnJ6 revealed that it interacts with oxidoreductases, photosynthetic proteins (mainly PSI), and proteases. Our in vivo experiments with Chlamydomonas reinhardtii insertional mutants (ΔZnJ6) expressing a low level of ZnJ6, suggested that the mutant is more tolerant to oxidative stress. In contrast, the wild type has better protection at elevated temperature and DTT induced stress. We propose that DnaJ-like chaperone ZnJ6 assists in the prevention of protein aggregation, stress endurance, and maintenance of redox balance.One-sentence summary ZnJ6 is a redox-regulated DnaJ-like chaperone associated with the thylakoid membrane and involved in the prevention of protein aggregation and stress endurance.