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The use of mitochondrial metabolomics via combined GC/LC-MS profiling to reveal metabolic dysfunctions in sym1-deleted yeast cells
1, 2 , 3 , 3, 4 , * 1, 4
1  Center for Biological Systems Analysis ZBSA, Albert-Ludwigs-University Freiburg, 79104 Freiburg, Germany
2  Institute of Pharmaceutical Sciences, Albert-Ludwigs-University Freiburg, 79104 Freiburg, Germany
3  Institute of Biochemistry and Molecular Biology, ZBMZ, Faculty of Medicine, University of Freiburg, 79104 Freiburg, Germany
4  BIOSS Centre for Biological Signalling Studies, University of Freiburg, 79104 Freiburg, Germany


SYM1 is an ortholog of the human MPV17 gene whose mutation causes mitochondrial DNA depletion syndrome. Sym1 protein is located in the inner mitochondrial membrane and its deletion results in impaired mitochondrial bioenergetic functions and morphological features under stress conditions. However, the functions of both Mpv17 and Sym1 have not been clearly characterized. Recently, compartment-specific metabolic alterations to mitochondrial mutations or inhibitors were revealed by analyzing isolated mitochondria. This development opens new doors for uncovering the function of Sym1. In order to find evidence for the molecular function of Sym1, mitochondria and the corresponding cytoplasmic fraction were isolated from wild type and sym1Δ cells through differential centrifugation. The samples were subjected to GC-MS profiling, after derivatization, or analyzed directly by LC-MS profiling, without derivatization. Eighty-nine metabolites were annotated by GC-MS profiling, while forty-five were annotated by LC-MS profiling. TCA cycle intermediates were reduced overall in sym1Δ. This correlates with the results of Dallabona et al. which showed severe OXPHOS defects of sym1Δ under stress conditions. At the same time, reduced glutathione was accumulated in mitochondria but reduced in cytosol, indicating an impaired redox balance in mutant cells. Interestingly, glutamine and aspartate, which can feed the TCA cycle, were up-regulated or maintained in mitochondria of sym1Δ. Furthermore, saccharopine was up-regulated while lysine was down-regulated in sym1Δ, exposing arrested lysine biosynthesis. Overall, GC- and LC-MS profiling in one workflow complement each other in identifying metabolites, which is helpful in understanding the metabolic dysregulations caused by deletion of sym1.

Keywords: SYM1; metabolic profiling; GC-MS; LC-MS