Fig. 3.

Preferred and less-favored nitrogen sources elicit congruent P-S6 and global growth responses.

A. Wild-type SC5314 cells were starved for nitrogen by growing in YNB containing glucose (2%) without ammonium sulfate (“-”) for 1 hour at 30°C; as a control, cells were grown in medium supplemented with ammonium sulfate (38 mM) (“+”). After 1 hour, ammonium sulfate was added to one culture of nitrogen-starved cells to a final concentration of 38 mM (“Add back “+””) and water was added to a second culture (“Add back “-””) as a control; all cells were then grown at 30°C for 30 minutes. Cell lysates were probed for P-S6 and H3.

B. Wild-type SC5314 cells were grown at 30°C without shaking in YNB with glucose (2%) containing 100 mM MES Buffer at pH 5, supplemented with ammonium sulfate (38 mM) or one of the following amino acids: glutamine, arginine, leucine, glutamate, proline, tryptophan (10 mM); a control sample was grown in the absence of a nitrogen source. The OD₆₀₀ of the cells in the different culture conditions were monitored in 15-minute intervals for about 24 hours.

C. Exponentially-growing wild-type SC5314 cells were transferred to YNB containing glucose (2%) and supplemented with the indicated nitrogen sources: ammonium sulfate (5 or 38 mM), or one of the following amino acids glutamine, glutamate, proline, arginine, or tryptophan (10 mM); a culture was also set up in medium without a nitrogen source. All cultures were grown for 1 hour at 30°C and cell lysates were probed on separate blots for P-S6 (top) and total S6 (bottom); for the loading control, anti-tubulin antibody was used on both blots.

D. Exponentially-growing wild-type JKC1361 and isogenic TOR1 heterozygous (tor1/TOR1) JKC1345 strains were transferred to YNB containing glucose (2%) and supplemented with the indicated nitrogen sources: ammonium sulfate (38 mM) or proline (5 or 10 mM) or serine (5 or 10 mM) and grown for 1 hour at 30°C. Cell lysates were probed for P-S6 and tubulin.

E. Exponentially-growing wild-type JKC317 and isogenic sch9 deletion (sch9-/-) CCS3 strains were transferred to YNB containing glucose (2%) and supplemented with the indicated nitrogen sources: ammonium sulfate (5 mM) or one of the following amino acids proline, leucine, or glutamine (10 mM); cultures were also set up in medium containing no nitrogen source. All cultures were grown for 1 hour at 30°C and cell lysates were probed for P-S6 and tubulin.