Functional relationships of three NFU proteins in the biogenesis of chloroplastic iron-sulfur clusters

Iron-sulfur clusters are required in a variety of biological processes. Biogenesis of iron-sulfur clusters includes assembly of iron-sulfur clusters on scaffold complexes and transfer of iron-sulfur clusters to recipient apoproteins by iron-sulfur carriers, such as nitrogen-fixation-subunit-U (NFU)-type proteins. Arabidopsis thaliana has three plastid-targeted NFUs: NFU1, NFU2, and NFU3. We previously discovered that nfu2 ^-/- nfu3 ^-/- mutants are embryo lethal. The lack of viable nfu2 ^-/- nfu3 ^-/- mutants posed a serious challenge. To overcome this problem, we characterized nfu2-1 ^-/- nfu3-2 and nfu2-1 ^-/- sesquimutants. Simultaneous loss-of-function mutations in NFU2 and NFU3 have an additive effect on the declines of 4Fe-4S-containing PSI core subunits. Consequently, the sesquimutants had much lower PSI and PSII activities, much less chlorophyll, and much smaller plant sizes, than nfu2-1 and nfu3-2 single mutants. These observations are consistent with proposed roles of NFU3 and NFU2 in the biogenesis of chloroplastic 4Fe-4S. By performing spectroscopic and in vitro reconstitution experiments, we found that NFU1 may act as a carrier for chloroplastic 4Fe-4S and 3Fe-4S clusters. In line with this hypothesis, loss-of-function mutations in NFU1 resulted in significant declines in 4Fe-4S- and 3Fe-4S-containing chloroplastic proteins. The declines of PSI activity and 4Fe-4S-containing PSI core subunits in nfu1 mutants indicate that PSI is the main target of NFU1 action. The reductions in 4Fe-4S-containing PSI core proteins and PSI activity in nfu3-2, nfu2-1, and nfu1 single mutants suggest that all three plastid-targeted NFU proteins contribute to the biogenesis of chloroplastic 4Fe-4S clusters. Although different insertion sites of T-DNA lines may cause variations in phenotypic results, mutation severity could be an indicator of the relative importance of the gene product. Our results are consistent with the hypothesis that NFU3 contributes more than NFU2 and NFU2 contributes more than NFU1 to the production of 4Fe-4S-containing PSI core subunits.