Functional Relationships of Two NFU Proteins in Maintaining the Abundances of Mitochondrial Iron–Sulfur Proteins

Iron-sulfur clusters are involved in many biological processes, including photosynthetic electron transport in the chloroplast and respiratory electron transport in the mitochondrion. Iron-sulfur cluster biosynthesis requires iron-sulfur carriers such as nitrogen-fixation-subunit-U [NFU]-type proteins. The <i>Arabidopsis thaliana</i> nuclear genome encodes two mitochondrion-targeted NFU proteins: NFU4 and NFU5, previously reported to have a primary role in the biosynthesis of the lipoate cofactor, mediated by the 4Fe-4S enzyme lipoyl synthase. Through in vitro reconstitution and spectroscopic analysis, we found that recombinant NFU4 and NFU5 proteins had UV-visible features characteristic of 4Fe-4S clusters. In addition, we confirmed that double homozygous, complete loss-of-function <i>nfu4 nfu5</i> mutants had an embryo-lethal phenotype. To investigate the functional relationship between NFU4 and NFU5, we generated sesquimutants that were homozygous loss-of-function for one gene and heterozygous for the other, which appeared slightly smaller than <i>nfu4-2</i>, <i>nfu4-4</i>, and <i>nfu5-1</i> single mutants. This suggests that the simultaneous decrease in levels of NFU4 and NFU5 proteins may have an additive effect on plant growth. Quantitative reverse transcription PCR showed that the <i>NFU4</i> transcript was absent in mutants homozygous for <i>nfu4-2</i> and <i>nfu4-4</i> and that the <i>NFU5</i> transcript level was substantially reduced in the <i>nfu5-1</i> single mutant or sesquimutants. Consistent with the transcript data, the abundances of NFU4 and NFU5 proteins were either virtually absent or substantially reduced in the corresponding single mutants and sesquimutants. Immunoblot analysis showed that most <i>nfu4</i> and <i>nfu5-1</i> single, double, and sesquimutants had significant reductions in the levels of mitochondrial 4Fe-4S proteins, such as aconitase (ACO) and biotin synthase 2 (BIO2; note that BIO2 also contains a 2Fe-2S cluster). In addition, <i>nfu4 nfu5</i> sesquimutants showed substantial reductions in the protein level of the 75-kDa subunit of respiratory complex I (CI75), which contains one 2Fe-2S cluster and two 4Fe-4S clusters. These observations indicate that NFU4 and NFU5 are important in maintaining the levels of mitochondrial 4Fe-4S proteins. Such observations are also consistent with the hypothesis that NFU4 and NFU5 may serve as iron-sulfur carriers and may play a role in the transfer of 4Fe-4S clusters to recipient apoproteins, such as ACO and CI75, during the biogenesis and maturation of mitochondrial 4Fe-4S clusters.