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+/‐ nfu3‐2−/− 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.

Menkes syndrome is an inherited disorder that affects copper levels in the body because of a mutation in the enzyme ATP7A. This protein is found in all cells throughout the body, except liver cells, and is essential for shuttling copper between two cellular locations. ATP7A can be found within the Golgi apparatus to supply copper to certain enzymes, but if copper levels are elevated in the cells environment, it can move to the cell membrane and shuttle copper out of the cell. When this protein is mutated, copper becomes poorly distributed throughout the cells of the body and can lead to reduced activity of many copper containing enzymes. This leads to brain abnormalities, bone weakness, degeneration of retina, and slowed development and growth. However, effects on the immune system and specifically macrophages are still unclear. These cells can become stimulated by foreign pathogens and secrete a cytokine, called Interferon‐gamma (IFN‐γ), which activates and recruits surrounding immune cells for an immune response. IFN‐γ stimulates production of reactive oxygen species (ROS) from metals within the cell to kill pathogens after phagocytosis. Due to its capability to aid in an immune response, it is hypothesized that IFN‐γ coupled with copper—a producer of reactive oxygen species—would activate macrophages and improve the cell's bactericidal effects. However, if ATP7A is mutated, and copper cannot be transported into the cell, its capacity to kill will decrease. Before experimentally determining the effects of IFN‐γ and copper on the immune system, the cytokine itself was synthesized. IFN‐γ encoding cDNA was amplified using PCR, and then annealed to the pET‐32‐Xa/LIC vector. The newly formed vector was successfully produced as determined by restriction endonuclease digestion, and then transformed into BL21‐DE3 E. coli. Induction of protein expression using IPTG was completed and visualized using SDS‐PAGE. IFN‐γ was isolated and purified using a HisTrap Nickel affinity column, and a SDS‐PAGE showed two bands, at 39 kDa (IFN‐γ) and 22 kDa (contaminated band). These results suggest a lack of purity due to an absence of protease inhibitors in solution, or low binding affinity to the nickel affinity column. Ongoing studies are observing macrophage phagocytosis and killing of bacteria in the presence and absence of IFN‐γ and copper to further understand the impact of Menkes syndrome on the immune system.Support or Funding InformationFunded by The Giving Well Family Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.