Figure 8.

Cells bearing the deregulated MTHFR do not show major changes in relative metabolic fluxes. A, yeast transformants of MET13_WT or MET13_R357A in met13Δmet15Δ (ABC2613) that were grown overnight in minimal medium with amino acid supplements and GSH were analyzed for growth. These were then reinoculated at 0.15 A₆₀₀ in fresh SD medium without any sulfur source. Methionine was added to the transformants after 3 h of secondary inoculation. The experiment was repeated three times, and a representative data set is shown. B, the ¹³C label redistribution via glycolysis, pentose phosphate pathway, TCA cycle, one-carbon metabolism (represented along with folate cycle, methionine cycle, methionine salvage pathway, polyamine synthesis, transsulfuration pathway, and amino acid biosynthesis are highlighted. Also, the fate of externally fed, histidine, lysine, leucine, and methionine is depicted. The proportional MIDs of protein-derived amino acid fragments retrobiosynthetically report on the labeling of precursors. These amino acid fragments were obtained by TBDMS derivatization and GC-MS ionization and are presented by their m/z and carbon backbone. The ¹³C redistribution in the carbon atoms of central metabolites is derived from our analysis and highlighted via filled light-gray circles. The open circles represent unlabeled carbon, whereas the circles with close vertical lines represents either unlabeled ¹²C or labeled ¹³C incorporation. In the methionine cycle and methionine salvage pathway, additional carbons from ATP (adenine and ribose unit) are depicted in circles with close vertical lines. An asterisk represents significance at 99% significance level analyzed via t test. Filled gray circles, ¹³C; white circles, labeled carbon; circles with close vertical lines, unlabeled ¹²C or labeled ¹³C; PPP, pentose phosphate pathway; G6P, glucose-6-phosphate; 3PG, 3-phosphoglycerate; DHF, dihydrofolate; THF, tetrahydrofolate; 5,10-CH₂THF, 5,10-methylene tetrahydrofolate; 5-CH₃THF, 5-methyl tetrahydrofolate; 5-HCO-THF, 5- formyltetrahydrofolate; 10-HCOTHF, 10-formyltetrahydrofolate; 5,10-CH*THF, 5,10-methenyl tetrahydrofolate; dcSAM, decarboxylated SAM; MTA, methylthioadenosine; MTR, methylthioribose; SHMT, serine hydroxymethyl transferase; TYMS, thymidylate synthase; GART, glycinamide ribonucleotide transformylase; AICART, aminoimidazolecarboxamide ribonucleotide formyltransferase.