FIGURE 6:

The PPP-GDH-PLP nodal enzymes are methionine dependent and largely GCN4 dependent. (A) Snz1p, Gnd2p, and Gdh1p amounts in WT or gcn4Δ cells, with methionine in the medium as the variable. WT and gcn4Δ cells expressing FLAG-tagged Snz1p or Gnd2p or Gdh1p were shifted from RM to MM or MM plus methionine, and amounts of these proteins were detected by Western blotting. A representative blot is shown in each case (n = 2). Also see Supplemental Figure 10. (B) NADP-dependent glutamate dehydrogenase activity with methionine in the medium as a variable. Crude extracts of WT cells grown in RM and shifted to MM or MM plus methionine were analyzed for intracellular biosynthetic NADP–glutamate dehydrogenase activity (n = 4). (C) Relative nucleotide amounts in the presence of methionine in WT or gcn4Δ cells. WT and gcn4Δ cells grown in RM were shifted to MM (4 h) with and without methionine, and the relative amounts of AMP and guanosine 5’-monophosphate (GMP) from metabolite extracts of the respective samples were measured by LC-MS/MS (n = 2 biological replicates, with technical replicates). In all panels, data indicate mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.