The chloroplastic NFU1 maturation factor sustains iron-sulfur cluster assembly in the dark in Chlamydomonas.

The sulfur mobilization (SUF) machinery is required for the synthesis of iron-sulfur (Fe-S) clusters and their insertion into client proteins in plastids. The final step relies on several Fe-S cluster transfer proteins, including NifU-like (NFU) proteins. In this study, we focused on the chloroplastic NFU1 from the green microalga Chlamydomonas reinhardtii. It possesses an N-terminal putative endonuclease domain that is absent in orthologs from angiosperms. Using a reverse genetic approach, we demonstrated that NFU1 serves as the major maturation factor for several [4Fe-4S]-cluster containing proteins involved in specialized pathways. This includes an atypical hybrid cluster protein, the pyruvate-ferredoxin oxidoreductase and an iron-iron hydrogenase operating in anoxia, as well as the dark-operative protochlorophyllide a oxidoreductase (DPOR) involved in chlorophyll synthesis in the dark. Based on the decreased abundance of chloroplast ribosomal proteins observed by proteomics in the nfu1 mutants, the lack of endonuclease activity of the N-terminal domain and the fact that NFU1 was previously reported as associated with chloroplast ribosomes, we propose that NFU1 contributes to the co-translational insertion of the [4Fe-4S] cluster(s) in the chloroplast-encoded DPOR subunits. The strong co-occurrence between genes encoding elongated NFU1 representatives and DPOR subunits in the green lineage supports this intertwined function and the importance of the N-terminal domain for NFU1 function.