Mitochondria have played a major role in the evolution of eukaryotic cells, in no small part due to their role in energy metabolism. The mitochondrial inner membrane houses the respiratory chain, a series of five membrane-bound complexes that work together to supply the respiring cell with the majority of its energy needs. The first of these complexes is known as NADH: Ubiquinone Oxidoreductase (Complex I) and is composed of 45 subunits. The coordinated assembly of such a large complex requires a number of chaperones known as assembly factors which promote the biogenesis of the complete holoenzyme. Understanding the molecular function of these assembly factors is important as mutations in them result in complex I deficiency and metabolic disease.

Complex I is unique in its absence from the respiratory chains of several unicellular eukaryotic organisms. Recent phylogenetic analysis has revealed several candidate assembly factors by virtue of the loss of their genes concurrent to those encoding for genuine complex I subunits. Here, we utilise a strategy for the generation of targeted gene knock-outs of candidate complex I assembly factors in tissue culture cell lines using Transcription activator-like effector nuclease (TALEN) technology. This work aims to both validate and also develop a detailed molecular understanding of the roles these assembly factors play in complex I biogenesis.