Kıvanç Birsoy, Tim Wang, Walter W. Chen, Elizaveta Freinkman, Monther Abu-Remaileh, David M. Sabatini
When mitochondrial respiration is inhibited, it is observed that a cell's ability to proliferate is diminished. It is also known that cells with inhibited oxidative phosphorylation (OXPHOS) are still able to proliferate, if cultured in high concentrations of pyruvate (a metabolite which is the by-product of glycolysis, and feeds the tricarboxylic acid cycle). In this study, the authors screen a library of ~3,000 metabolic enzymes, treated with a low dose of complex I inhibitor phenformin, whose loss causes a severe anti-proliferative phenotype. This would reveal genes which are required in an adaptive response to mild OXPHOS inhibition.
The best scoring gene in their screen was GOT1, a component of the malate-aspartate shuttle, which transfers substrates for OXPHOS into the mitochondrial matrix. They find that GOT1-null cells are hypersensitive to phenformin, causing their proliferation to halt, at doses where wild-type cells do not.
Under normal conditions, GOT1 shuttles the amino acid aspartate, into the mitochondrial matrix. They find that, upon inhibition of complex I, GOT1 reverses its flux and exports aspartate. Aspartate is an amino acid, required for the synthesis of proteins, purines and pyramidines. Normally, it is generated in the mitochondrial matrix; the authors propose that during ETC inhibition, a drop in NAD+/NADH ratio causes aspartate synthesis to shut down, using normal pathways. Thus GOT1 reverses its flux, to become a source of this amino acid.
It is also known that supplementation of pyruvate can overcome the inhibitory effects of several ETC inhibitors. By culturing GOT1-null cells with/without pyruvate, the authors show that there is no benefit to supplementing cells with pyruvate under ETC inhibition, if cells lack GOT1. Thus a key mechanism of pyruvate supplementation is GOT1-catalyzed aspartate synthesis. Interestingly, these conclusions appear to hold in cybrid cell lines harbouring mtDNA mutations (homoplasmic mutations in CYTB and tRNA lysine were tested). When these cells overexpress an aspartate transporter SLC1A3, and cultured in high aspartate medium, their proliferation rate recovers to similar levels as wild-type cells.