Yeast cells respond to heat stress by remodeling their gene expression, resulting in the changes of the corresponding proteins and metabolites. Compared to the intensively investigated transcriptome and proteome, the metabolic response to heat stress is not sufficiently characterized. Mitochondria have been recognized to play an essential role in heat stress tolerance. Given the compartmentalization of the cell, it is not clear if the heat stress-induced metabolic response occurs in mitochondria or in the cytosol. Therefore, a compartment-specific metabolite analysis was performed to analyze the heat stress-induced metabolic response in mitochondria and the cytoplasm. In this work, the isolated mitochondria and the cytoplasm of yeast cells grown at permissive temperature and cells adapting to heat stress were subjected to mass spectrometry-based metabolomics. Over a hundred metabolites could be identified, covering amino acid metabolism, energy metabolism, arginine metabolism, purine and pyrimidine metabolism, and others. Highly accumulated citrulline and reduced arginine suggested remodeled arginine metabolism. A stable isotope-labeled experiment was performed to analyze the heat stress-induced metabolic remodeling of the arginine metabolism, identifying activated de novo ornithine biosynthesis to support arginine and spermidine synthesis. The short-term increased spermidine and trehalose suggest their important roles as heat stress markers. These data provide metabolic clues of heat stress-induced metabolic remodeling, which helps in understanding the heat stress response.

Introduction Subcellular compartmentalization enables eukaryotic cells to carry out different reactions at the same time, resulting in different metabolite pools in the subcellular compartments. Thus, mutations affecting the mitochondrial energy metabolism could cause different metabolic alterations in mitochondria compared to the cytoplasm. Given that the metabolite pool in the cytosol is larger than that of other subcellular compartments, metabolic profiling of total cells could miss these compartment-specific metabolic alterations. Objectives To reveal compartment-specific metabolic differences, mitochondria and the cytoplasmic fraction of baker’s yeast Saccharomyces cerevisiae were isolated and subjected to metabolic profiling. Methods Mitochondria were isolated through differential centrifugation and were analyzed together with the remaining cytoplasm by gas chromatography–mass spectrometry (GC–MS) based metabolic profiling. Results Seventy-two metabolites were identified, of which eight were found exclusively in mitochondria and sixteen exclusively in the cytoplasm. Based on the metabolic signature of mitochondria and of the cytoplasm, mutants of the succinate dehydrogenase (respiratory chain complex II) and of the F_OF₁-ATP-synthase (complex V) can be discriminated in both compartments by principal component analysis from wild-type and each other. These mitochondrial oxidative phosphorylation machinery mutants altered not only citric acid cycle related metabolites but also amino acids, fatty acids, purine and pyrimidine intermediates and others. Conclusion By applying metabolomics to isolated mitochondria and the corresponding cytoplasm, compartment-specific metabolic signatures can be identified. This subcellular metabolomics analysis is a powerful tool to study the molecular mechanism of compartment-specific metabolic homeostasis in response to mutations affecting the mitochondrial metabolism.

XD14 is a 4-acyl pyrrole derivative, which was discovered by a high-throughput virtual screening experiment. XD14 inhibits bromodomain and extra-terminal domain (BET) proteins (BRD2, BRD3, BRD4 and BRDT) and consequently suppresses cell proliferation. In this study, metabolic profiling reveals the molecular effects in the human breast cancer cell line MCF-7 (Michigan Cancer Foundation-7) treated by XD14. A three-day time series experiment with two concentrations of XD14 was performed. Gas chromatography-mass spectrometry (GC-MS) was applied for untargeted profiling of treated and non-treated MCF-7 cells. The gained data sets were evaluated by several statistical methods: analysis of variance (ANOVA), clustering analysis, principle component analysis (PCA), and partial least squares discriminant analysis (PLS-DA). Cell proliferation was strongly inhibited by treatment with 50 µM XD14. Samples could be discriminated by time and XD14 concentration using PLS-DA. From the 117 identified metabolites, 67 were significantly altered after XD14 treatment. These metabolites include amino acids, fatty acids, Krebs cycle and glycolysis intermediates, as well as compounds of purine and pyrimidine metabolism. This massive intervention in energy metabolism and the lack of available nucleotides could explain the decreased proliferation rate of the cancer cells.

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.

Mutations in mitochondrial membrane proteins could cause physiological and metabolic alterations in mitochondria as well as in cytosol. In order to address the origin of these alterations, mitochondria and cytosol of yeast wild-type BY4741 and two mutants, sdh2Δ and atp4Δ, were isolated from whole cells. These three compartments, namely mitochondria, cytosol and whole cell, were analyzed by gas chromatography-mass spectrometry based metabolic profiling, identifying seventy-three metabolites altogether, from which sixteen or ten were not detected either in mitochondria or cytosol. Compartment-specific distribution and regulation of metabolites were observed, showing the responses to the deletions of sdh2 and atp4. Based on the metabolic signature in mitochondrial matrix and cytosol, both mutants can be discriminated from wild-type by principal component analysis. De letions of electron chain transport components, sdh2 and atp4, altered not only citrate cycle related metabolites, but also diverse metabolites including amino acids, fatty acids, purine and pyrimidine intermediates and others. By applying metabolomics to isolated mitochondria and cytosol, compartment-specific metabolic regulation can be identified, which is helpful in understanding the molecular mechanism of mitochondrial homeostasis in response to genetic mutations.