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Chen, Walter W.; Freinkman, Elizaveta; Wang, Tim; Birsoy, Kıvanç; Sabatini, David M.
Cell, 08/2016, Letnik: 166, Številka: 5Journal Article
Mitochondria house metabolic pathways that impact most aspects of cellular physiology. While metabolite profiling by mass spectrometry is widely applied at the whole-cell level, it is not routinely possible to measure the concentrations of small molecules in mammalian organelles. We describe a method for the rapid and specific isolation of mitochondria and use it in tandem with a database of predicted mitochondrial metabolites (“MITObolome”) to measure the matrix concentrations of more than 100 metabolites across various states of respiratory chain (RC) function. Disruption of the RC reveals extensive compartmentalization of mitochondrial metabolism and signatures unique to the inhibition of each RC complex. Pyruvate enables the proliferation of RC-deficient cells but has surprisingly limited effects on matrix contents. Interestingly, despite failing to restore matrix NADH/NAD balance, pyruvate does increase aspartate, likely through the exchange of matrix glutamate for cytosolic aspartate. We demonstrate the value of mitochondrial metabolite profiling and describe a strategy applicable to other organelles. Display omitted •A workflow for absolute quantification of mitochondrial matrix metabolites•Rapid and specific isolation of mitochondria from cells for metabolite profiling•Profiling guided by MITObolome, a set of all predicted mitochondrial metabolites•Dynamics of mitochondrial metabolism revealed by quantification of >100 metabolites Metabolite profiling of intact mammalian mitochondria captures dynamics of mitochondrial metabolism not revealed by whole-cell analysis.
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