AbstractsBiology & Animal Science

The main function of mitochondria is the generation of cellular energy through the oxidative phosphorylation (OXPHOS) pathway. Calcium homeostasis, apoptotic signaling, the citric acid and urea cycles, iron sulfur cluster, steroid and heme synthesis are other biosynthetic pathways that take place within mitochondria, making this organelle indispensable for the proper function of the cell. Mitochondrial disorders have an incidence of at least 1 in 5000 live births and they range from neonatal fatalities to late-onset neurodegeneration. Mammalian mitochondria have a circular genome reminiscent of their prokaryotic origins. It is organized into protein/DNA complexes called nucleoids, which are necessary for its stability, expression and segregation. Mitochondrial DNA (mtDNA) encodes 13 subunits of the OXPHOS complexes I, III, IV and V, 22 tRNAs and two rRNAs.Complex I (NADH ubiquinone oxidoreductase) is the first complex of the OXPHOS pathway. It is responsible for the oxidization of NADH and for the pumping of protons from the mitochondrial matrix to the intermembrane space, in this way contributing to the formation of a proton gradient that is ultimately used to synthesize ATP. Complex I deficiency is the most common cause of mitochondrial disease in infants. Mutational analyses have identified defects in several of the structural components of the complex, however these mutations explain only 50% of the cases, implicating nuclear-encoded chaperones or assembly factors as an important cause of disease. In this study we demonstrate that complex I biogenesis is nucleated by an early subcomplex of 315 kDa containing at least the nuclear-encoded subunit NDUFS2 and the mtDNA-encoded subunit ND1. The assembly factors NDUFAF3, NDUFAF4, NDUFAF7, C8orf38 and, C20orf7 are necessary for the assembly and stabilization of the 315 kDa intermediate. By using RNAi technology to knock-down the expression of NDUFAF2, NDUFAF3, NDUFAF4, C8orf38, C20orf7 and, NDUFAF7 we demonstrate that early complex I assembly defects result in the proteolytic degradation of the ND1 subunit by the inner membrane protease m-AAA AFG3L2, in this manner regulating the latter steps of the assembly pathway. We performed an in depth functional characterization of the NDUFAF7 assembly factor and demonstrated that it is responsible for the symmetric dimethylation of Arg85 of the NDUFS2 subunit after it assembles into complex I, stabilizing an early assembly intermediate.We showed that the AAA+ LONP1 protease, which is part of the protein quality control system of the mitochondrial matrix, plays an essential role in the maintenance and expression of the mitochondrial genome. LONP1 depletion selectively impairs the degradation and processing of the mitochondrial targeting sequence of the nucleoid components, SSBP1 and MTERFD3, the RNA granule protein, FASTKD2 and, the matrix protease, CLPX. Likewise, LONP1 knock-down caused the accumulation of protein aggregates primarily containing soluble DNA/RNA Associated Proteins (DRAPs) and mitochondrial ribosomal structural…