The proxiomes of CDJ5 and PGRL1 in Chlamydomonas reinhardtii overlap depending on the ability of CDJ5 to bind a 4Fe-4S cluster

The chloroplast chaperone HSP70B from Chlamydomonas reinhardtii works with the J-domain co-chaperones CDJ1 to CDJ6. CDJ1 delivers unfolded proteins, while CDJ2 delivers VIPP1 as substrate to HSP70B. CDJ3 to CDJ5 contain 4Fe-4S clusters in addition to the J domain, but their function is unknown. To investigate the function of CDJ5, we performed TurboID-mediated proximity labeling on wild-type CDJ5 (CDJ5-WT) and CDJ5 mutants with an impaired ability to stimulate HSP70B's ATPase activity (CDJ5-AAA) or bind a 4Fe-4S cluster (CDJ5-SSS). Our results revealed that the proxiomes of all CDJ5 variants contained HSP70B and HSP90C. Furthermore, the proxiomes of CDJ5-WT and CDJ5-AAA overlapped extensively but differed from that of CDJ5-SSS, suggesting that the localization of CDJ5 to a chloroplast microcompartment depends on the presence of a functional 4Fe-4S cluster or its redox state. The CDJ5-WT and CDJ5-AAA proxiomes were enriched with proteins that regulate photosynthetic electron flow or are involved in the biogenesis of thylakoid membrane protein complexes and pigments. These proteins were also present in the proxiome of PGRL1 found in the CDJ5-WT and CDJ5-AAA proxiomes. Overall, our results suggest that CDJ5 acts with HSP70B/HSP90C via its 4Fe-4S cluster to regulate photosynthetic electron flow and thylakoid membrane protein complex biogenesis.