Mass spectrometry-based metabolomics : convergence of Snf1/AMPK and methionine metabolism to control mitochondrial respiration

Mass spectrometry-based metabolomics has become increasingly popular in biology and molecular medicine. Analysis of the key metabolites in biological fluids has become an important part of improving disease diagnosis. In this context, chromatography coupled with mass spectrometry represents a valid tool to characterize and quantify numerous compounds present in biological matrices. In the present work, we have explored the role of methionine metabolism on cellular functions of a prototrophic yeast strain during exponential growth phase and determining how this affects the metabolic state upon SNF1 deletion. Experimental Yeast strains and growth conditions S. cerevisiae strains used in this study are reported in Table 1. Synthetic medium (SD) contains 2% glucose, 6.7 g/L of Yeast Nitrogen Base (Difco). Methionine was added to the concentrations indicated in figure legends. In these conditions, cells exhibit exponential growth between OD600nm = 0.1 per ml (equivalent to 2*106 cells/ml) and OD600nm = 2.5 per ml (5*107 cells/ml); all experiments were performed in exponential phase of growth (OD600nm/ml 0.5-1) or post-log phase (24 h, 48 h and 72 h after the beginning of exponential phase). Antimycin A was added to a final concentration of 1 μg/ml from 2 mg/ml stock in 100% ethanol; the same volume of ethanol was added in the control culture. Cells were properly manupulated in order to extract RNA and carried out all the necessary biological investigation in addition to metabolites analysis by GC-MS. Metabolites analysis 13C-labelling of proteinogenic amino acids was achieved by growth on 20 g/L glucose as a mixture of 80% (w/w) unlabelled and 20% (w/w) uniformly labeled [U-13C]glucose (13C, 99 %; Cambridge Isotope Laboratories, Inc). Cells from an overnight minimal medium culture were washed and used for inoculation below an OD600 of 0.03. 13C-labelled biomass aliquots were harvested by centrifugation during the mid-exponential growth phase at an OD600 of ≤1. The cells (about 0.3 mg of dry biomass) were washed once with sterile water, and hydrolysed in 150 μL 6 M HCl at 105 ºC for 6 h. The hydrolysate was dried in a heating block at 80 ºC under a constant airflow. Before the GC/MS analyses all samples were subjected to a derivatization step as follows. Each sample was resuspended in 30 μL of acetonitrile, followed by 30 μL of MBDSTFA (N-methyl-N-ter-butyldimethylsilyl-trifluoroacetamide). The resulting mixture, contained in a closed vial, was stirred for 10 min and centrifuged for 15 sec. Then, the vial was incubated at 85° C. After 1 h, the sample slowly reached room temperature and was analysed by GC-MS (single quadrupole). A METAFOR (metabolic flux ratio) analysis was perfomed: the mass isotopomer distribution of proteinogenic amino acids was used to calculate the split ratios of key branching points of yeast central metabolism using the software FIAT FLUX [3]. The results show that methionine addition leads to Snf1 and Acc1 phosphorylations as well as a general slow-down of proliferation, boosting mtDNA synthesis, mitochondrial pyruvate uptake and TCA cycle activity. Remarkably, mitochondrial proteomic analysis highlight that methionine upregulates proteins of the pyruvate dehydrogenase complex, TCA cycle and respiratory chain complexes. Thus, Snf1/AMPK and methionine metabolism converge to control mitochondrial respiration in glucose repressed conditions. References: [1] Kushnirov, V. V. (2000). Yeast 16, 857–860. [2] Pessina, S., Tsiarentsyeva, V., Busnelli, S., Vanoni, M., Alberghina, L., and Coccetti, P. (2010). Cell Cycle 9, 2189–2200. [3] Zamboni, N., Fischer, E., and Sauer, U. (2005). BMC Bioinformatics 6, 209.