Role of MP68 in mitochondrial complex V biogenesis and function

Submission note: A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy to the Department of Biochemistry and Genetics, School of Molecular Sciences, College of Science, Health and Engineering, La Trobe University, Bundoora, Victoria.

Mitochondria represent the main source of energy of the cell generating ATP through the oxidative phosphorylation (OXPHOS). ATP is synthetised by F1-Fo ATPase (complex V) that uses the electrochemical gradient generated by the mitochondrial respiratory chain complexes to phosphorylate ADP. Many other roles have been proposed for complex V from maintaining cristae morphology to mitochondrial permeability transition. In the absence of mitochondrial membrane potential complex V can also hydrolyse ATP to pump H+ from the matrix in the inter membrane space pointing out how important its regulation can be for cell growth and survival. Both nuclear and mitochondrial genomes contribute to the biogenesis of this sophisticated molecular machinery; although the assembly, structure and function of the matrix part have been well understood in the past years, very little is known about the membrane part and its functions. 6.8 kDa Mitochondrial Proteolipid (MP68) is a small hydrophobic inner membrane protein that has been recently identified as a subunit of the membrane part of the vertebrate ATP synthase. I used CRISPR Cas9-mediated genome editing technology to investigate the role of this protein in F1-Fo ATPase assembly and function. I showed that MP68 knock out (KO) causes an increase in turnover of the membrane part of complex V as well as a defect in the assembly of the holocomplex with accumulation of various assembly intermediates in HEK293T cells. We also showed an increase in ATPase activity of isolated complex V suggesting a role of MP68 as part of the inhibitory mechanism that prevents the complex from hydrolysing ATP.