Alternative oxidase and its genetic, biochemical and physical interactions with the mitochondrial electron transport system of Drosophila melanogaster

Abstract

The mitochondrial alternative oxidase (AOX) is naturally absent in vertebrates and insects, but present in tunicates like Ciona intestinalis. The xenotopic expression of C. intestinalis AOX in Drosophila melanogaster and mice has shown promising results for the combat of mitochondrial dysfunction, exhibiting a potential therapeutic use in the future. AOX is a mitochondrial inner membrane enzyme that can bypass complexes III and IV of the respiratory chain, providing an additional pathway for oxygen reduction and coenzyme Q reoxidation, and allowing continued metabolic flux in situations where the electron transfer system is compromised. Preliminary leak respiration results using AOX-expressing larvae demonstrate possible formation of parallel electron transfer pathways in oxidative phosphorylation, through the use of supercomplexes (supramolecular organizations of mitochondrial complexes) and via mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH) with AOX. Interestingly, different species of Ciona do not have the genes that encode the subunits important for intercomplex interactions, suggesting that Ciona species may not have supercomplexes. If Ciona AOX evolved to function in a supercomplex-free mitochondrial inner membrane, this may explain why AOX seems to preferentially function in conjunction with non-proton pumping dehydrogenases, such as mGPDH. For the development of the project, we will use blue native polyacrylamide gel electrophoresis (BN-PAGE) combined with in-gel enzymatic activities or western blots, to quantitatively investigate the patterns of supercomplex formation in Drosophila larvae, linking the structural arrangement of the respiratory chain with functional and genetic descriptions of the interactions between mGPDH and AOX in lines with high and low AOX expression, and lines that combine AOX expression with varying levels of mGPDH. Additionally, we will use BN-PAGE in Ciona tissues to verify if Ciona does possess mitochondrial supercomplexes. (AU)