Mitochondrial Metabolomics of Sym1-Depleted Yeast Cells Revealed Them to Be Lysine Auxotroph

**Background:** Subcellular compartmentalization allows spatial distribution of metabolites, and compartment-specific metabolomics can reveal metabolic alterations that whole-cell analyses might miss. The yeast protein Sym1 is an ortholog of human MPV17, mutations in which cause mitochondrial DNA depletion syndrome (MDDS) characterized by hepatic and neurologic manifestations. Despite 98 reported pathogenic MPV17 variants by 2017, the protein's function remains undefined. Previous studies suggested Sym1 might be a transporter for TCA cycle intermediates or a channel-forming protein, but its precise role was unknown.

**Methods:** The authors used Saccharomyces cerevisiae sym1Δ deletion mutants and wild-type (WT) BY4741 controls grown in synthetic galactose medium. Mitochondria and cytoplasm were isolated separately. Three analytical approaches were combined: (1) untargeted GC–MS metabolic profiling, (2) targeted LC–MS for amino acids and pyrimidine intermediates, and (3) a novel U(H)PLC–QTOF/MS workflow focusing only on significantly changed metabolites. The QTOF workflow used HILIC chromatography with a 10-ppm mass accuracy and 0.25-min retention time window, followed by targeted MS/MS and library searching (METLIN). Statistical analyses included PCA, ANOVA with FDR correction, and heat map generation using MetaboAnalyst 5.0. Growth phenotype tests were performed on agar plates with/without specific metabolites at 19, 23, and 30 °C.

**Key Results:** PCA clearly discriminated cytoplasm from mitochondria (PC1) and sym1Δ from WT (PC2), with a greater shift between cytoplasm groups than mitochondria groups. ANOVA identified lysine as the top significantly changed metabolite. Lysine was significantly reduced in sym1Δ cells, while its biosynthesis intermediates 2-aminoadipate (2-AAA) and saccharopine were accumulated. Growth tests revealed that sym1Δ cells could not grow without lysine at any temperature tested (19, 23, and 30 °C), whereas arginine depletion only slightly repressed growth. Notably, addition of either lysine or its biosynthesis intermediates could not rescue the growth defect. Pyrimidine biosynthesis intermediates carbamoyl-aspartate and orotic acid were reduced in sym1Δ cells. The QTOF workflow identified 51 significantly changed metabolites total, of which 41 were novel compared to the combined GC–MS/targeted LC–MS method. Two metabolites—4-guanidinobutanal and 4-guanidinobutanoate from an alternative arginine degradation pathway—were identified for the first time in S. cerevisiae. Average mass deviations were 0.891 ppm (mitochondria) and 0.915 ppm (cytoplasm), with RT deviations of 0.00973 and 0.012 min respectively. Through library search, 7 and 8 metabolites were identified in mitochondria of WT and sym1Δ cells respectively, and 24 and 21 in cytoplasm of WT and sym1Δ cells respectively.

**Clinical Implications:** This study provides the first evidence linking Sym1/MPV17 dysfunction to lysine auxotrophy and pyrimidine metabolism alterations. The accumulation of saccharopine is particularly noteworthy, as prior research in C. elegans and mice showed that saccharopine accumulation causes mitochondrial dysfunction and liver failure—strikingly similar to the liver phenotype seen in 91% of MDDS patients with MPV17 mutations. The authors suggest that lysine metabolism may play a role in human MDDS pathophysiology, warranting future investigation. The reduced arginine and increased citrulline in sym1Δ cells resemble heat stress-induced metabolic remodeling, suggesting the mutant cells experience cellular stress even at normal growth temperatures, which may explain their inability to survive at 37 °C. The novel QTOF workflow successfully reduced data complexity while enabling discovery of 41 novel metabolites, demonstrating a useful strategy for future subcellular metabolomics studies. However, the origin and biological function of the identified di- and tripeptides remain unknown, and further studies under various stress conditions are needed to clarify Sym1's role in cellular stress management.