Functional analysis of the Fe-S cluster containing chloroplast J-domain proteins CDJ3-5

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Hsp70 chaperone systems are highly conserved and exist in the cytosol, the ER, mitochondria, and chloroplasts of eukaryotic cells. The chloroplast Hsp70 system is derived from the bacterial one and consists of an Hsp70, a GrpE-type nucleotide exchange factor, and so-called J-domain proteins. The latter bind specific substrate proteins and hand them over to Hsp70 for further processing. CDJ3-5 are three out of six J-domain proteins in the chloroplast of the unicellular green alga Chlamydomonas reinhardtii. In addition to the J domain and a domain of unknown function, they harbor a bacterial ferredoxin domain. For CDJ3 and 4 we could show that this domain binds a redox-active 4Fe-4S cluster. Members of the CDJ3-5 proteins exist in the chloroplasts of all Viridiplantae (plants and algae) as well as in the Thaumarchaeota, who have acquired it via horizontal gene transfer. We could show that CDJ3 and 4 interact ATP-dependently with chloroplast Hsp70 and stimulate its ATPase activity. However, they do not support the folding of denatured proteins and therefore appear not to play a role in protein homeostasis. About CDJ3 we know that its expression is inducible by light and that it forms a complex with RNA. CDJ3-5 all accumulate at very low levels. Nothing else is known about the function of CDJ3-5. The goal of this project is to shed light onto the functions of CDJ3-5 using the model system Chlamydomonas reinhardtii. With the help of a new toolkit for synthetic biology in Chlamydomonas, we want to generate constructs for the overexpression of CDJ3 wild-type and mutated forms. In the latter we will change the conserved HPD motif in the J domain into AAA in order to obtain dominant negative effects. These constructs will also code for C-terminal extensions that will allow us to i) identify stably interacting proteins via affinity purification and LC-MS/MS; ii) identify transient interaction partners via proximity labeling; iii) identify bound RNA species, also with the help of RIP-Chip and CLIP; iv) determine the suborganellar localization of CDJ3-5 in the chloroplast. Further, we want to employ the CRISPR/Cpf1 system to generate cdj3-5 knock-out mutants. These, existing cdj3/4 mutants, and the overexpressor lines will then be analyzed phenotypically. For this, we will first determine survival rates under diverse stress conditions. Next, we want to identify proteins differentially accumulating in the mutants compared to wild type by quantitative shotgun proteomics based on the 15N stable isotope. Finally, we want to analyze the Fe-S cluster in CDJ5 by Mösbauer spectroscopy to see, whether this cluster is distinct from those harbored by the phylogenetically more distant CDJ3/4 proteins.