Understanding the assembly of mitochondrial machines

Project Details

In humans, OXPHOS takes place on the five membrane protein complexes comprising the respiratory chain. These large multi-subunit complexes are together comprised of 93 subunits that are encoded by both mitochondrial (mt) and nuclear DNA. Mutations in all mtDNA protein coding genes and >180 nuclear genes impair OXPHOS and cause classical mitochondrial disease, however many of these genes do not encode subunits of the respiratory chain but rather proteins involved in OXPHOS biogenesis known as assembly factors. Although we have a good understanding of the enzymatic roles of each complex in OXPHOS, we still do not fully understand how the system is built and maintained, nor the specific roles of many subunits and assembly factors in this process.

We recently developed a novel systems biology approach, coupling CRISPR/Cas9 based gene-editing with quantitative proteomics tools, to profile the assembly pathways of OXPHOS complexes and discover new assembly factors (Stroud et al., Nature 2016). In addition to extensive gene-editing and quantitative proteomics tools, this project features classical molecular techniques such as mammalian cell culture, Blue-Native (BN) PAGE, western blotting, affinity enrichment techniques such as co-immunoprecipitation and BioID, cellular imaging, computational and structural biology, and metabolic measurements such as oxygen consumption and ATP production.