Multilayered regulation of TORC1 signaling by Ait1, Gcn2, and SEAC/GATOR during nitrogen limitation and starvation

Abstract

The Target of Rapamycin kinase Complex I (TORC1) is a central hub in the cell growth and metabolic control network of eukaryotes. How its upstream regulators cooperate to tune signaling across environmental conditions remains unclear. Here, we combine phosphoproteomics, TORC1 activity assays, and targeted genetic perturbations to dissect TORC1 regulation in Saccharomyces cerevisiae during transitions from a high-quality nitrogen source (glutamine) to a low-quality nitrogen source (proline), and on to complete nitrogen starvation. In proline medium, Ait1 and Gcn2 attenuate TORC1 activity, establishing a partially inhibited "Low Nitrogen Adaptive" state marked by extensive metabolic reprogramming without growth arrest. In contrast, during nitrogen starvation, SEAC, Ait1, and Gcn2 cooperate to drive TORC1 into a fully inhibited state, triggering widespread dephosphorylation of its downstream targets and entry into quiescence. Our results define a multilayered regulatory circuit that governs graded TORC1 control-a design likely conserved across eukaryotes.

Fig. 1. Sch9 phosphorylation during nitrogen limitation.

a Domains in Sch9, including the C2-domain, Kinase domain, Regulatory domain (RD), and C-terminal extension (CE) domain. TORC1 phosphorylates Sch9 at positions S711, T723, T737, S758, S765 (refs. ^,). Protein extracts were treated with NTCB prior to analysis, leading to cleavage after cysteine 555 (ref. ). b Anti-HA Western blot of protein extracts from wild-type cells expressing Sch9-3xHA from its native locus as they transition from growth in SD+Gln to growth in SD+Pro (top panel) or SD-N (bottom panel). The portion of the gel shown here only includes the ~30 kDa, C-terminal fragment of Sch9 containing the TORC1 target sites. c Quantification of Sch9 mobility data shown in (b) as well as 72 replicate experiments for SD+Pro and 8 replicate experiments for SD-N. Open circles and error bars show the average and standard deviation for each timepoint; filled circles show the data from individual experiments. SD+Pro samples were collected throughout this study as controls for experiments examining mutant strains (Figs. 5, 6). The standard error for all time-points in the SD+Pro dataset is less than 0.01. Statistical analysis (using a two-sided Welch’s t-test) comparing the two time-courses show that the Sch9 phosphorylation levels are similar in SD+Gln (p-value 6.36E-01) but diverge (p < 0.01) at subsequent timepoints.

Fig. 2. Global phosphorylation changes during nitrogen limitation.

a Heatmap of the 436 phosphopeptides that change significantly (>3-fold change, BH corrected p < 0.05) during the SD+Gln to SD+Pro transition as measured by mass spectrometry, and split into four groups using hierarchical clustering (n = 4, p-values from an ANOVA test). The full dataset is in Supplementary Data 1. b Line graphs for key proteins from (a) highlighting the timing and stability of the phosphorylation changes. Open circles and error bars show the average and standard deviation for each timepoint; filled circles show the data from individual experiments (n = 4). c, d Venn diagram showing the top Gene Ontology (GO) groups for the 232 proteins in panel (a), and a list of key target proteins. Asterisks mark proteins where a change in abundance accounts for the observed up- or down-regulation of one or more daughter phosphopeptide.

Fig. 3. Global phosphorylation changes during nitrogen starvation.

a Heatmap of the 630 phosphopeptides that change significantly (>3-fold change, p < 0.01, FDR < 3.2%) during the SD+Gln to SD-N transition, split into three groups to highlight variation in the timing of the response (n = 4, p-values from an ANOVA test). The full dataset is in Supplementary Data 2. b Venn diagram showing the top GO functional groups for the 460 proteins found in (a), and a list of key target proteins. c Scatterplot comparing the change in phosphopeptide abundance during the SD+Gln to SD+Pro and the SD+Gln to SD-N transition after 30 min, for all peptides that change significantly (>3-fold change, p < 0.01) in either condition. Solid and dashed lines show the values for a perfect correlation, and a spread of +/−0.5 on log₂ scale. The red dots highlight phosphopeptides that have a > 3-fold difference between conditions, and the parent proteins are listed below the graph.

Fig. 4. Global phosphorylation changes in nitrogen limitation versus nitrogen starvation.

a Scatterplot comparing phosphopeptide levels after four hours in SD-N and SD+Pro for all peptides that change significantly (>4-fold change, p < 0.001; FDR < 1%) in SD-N, SD+Pro, or rapamycin (3243 total). Each dot shows the mean abundance for a single phosphopeptide (n = 4). The solid and broken lines show the values for a perfect correlation and a spread of +/−0.5 on log₂ scale, respectively. Blue stars mark phosphopeptides that change >3-fold more in SD-N than in SD+Pro, and red stars mark those that change >3-fold more in both SD-N and rapamycin than in SD+Pro. The full dataset is in Supplementary Data 3. b Heatmap showing the data for all 286 phosphopeptides (on 185 proteins) marked by red stars in (a). (c) Raw data for representative proteins from (b). Open circles and error bars show the average and standard deviation for each timepoint; filled circles show the data from individual experiments (n = 4). d List of key proteins from (b).

Fig. 5. Role of Gtr1/2 and Pib2 in TORC1-Sch9 regulation during nitrogen limitation.

a, b Anti-HA Western blots of protein extracts from wild-type and mutant cells expressing Sch9-3xHA from its native locus as they transition from growth in SD+Gln to SD+Pro. Quantification of Sch9 mobility data shown in (a, b) as well as two repeat experiments for each mutant (n = 3) is shown on the right. The open circles and error bars show the average and standard deviation of the difference between the mutant and wild-type data, comparing samples grown, processed, and run together on the same gel (see Supplementary Table 2 for p-values). These values were added to the average wild-type data from Fig. 1 (shown by the broken line) for easy visualization. The filled circles show the difference calculated in each individual experiment.

Fig. 6. Role of Ait1, Gcn2, and Npr2 in TORC1-Sch9 regulation during nitrogen limitation.

a–c Anti-HA Western blots of protein extracts from wild-type and mutant cells expressing Sch9-3xHA from its native locus as they transition from growth in SD+Gln to SD+Pro. Quantification of Sch9 mobility data (n = 3) is shown on the right (see Supplementary Table 2 for p-values). Open circles and error bars show the average and standard deviation for each timepoint; filled circles show the data from individual experiments. The data were analyzed, and the graphs plotted, as described in Fig. 5.

Fig. 7. Contributions of Ait1+Gcn2 and Npr2 to TORC1 signaling during nitrogen limitation.

a Scatterplots comparing the phosphopeptide abundance change in wild-type, ait1Δgcn2Δ (left), and npr2Δ (right) strains subjected to nitrogen limitation (SD+Pro) for 30 min. Each dot shows the average data for a single phosphopeptide (n = 4). Peptides with strong regulation in only one mutant strain are highlighted, and the names of their parent proteins are listed below the graphs (with the number of distinct phosphosites in parentheses). The full dataset is in Supplementary Data 4. b Heatmap showing the significant phosphorylation changes (>3-fold change, p < 0.01, FDR < 3%) that occur after 30 min in SD+Pro, in wild-type, ait1Δgcn2Δ and npr2Δ mutant cells. The left columns show the log₂ fold change in each mutant caused by the SD+Gln to SD+Pro transition, the right columns show the ratio of the phosphopeptide abundance in mutant versus wildtype cells in SD+Gln medium. The bars show phosphopeptides that are hypo- (green) or hyper-phosphorylated (red) due to Npr2 activity and nutrient deprivation.

Fig. 8. Ait1, Gcn2, and Npr2 cooperate to regulate TORC1-Sch9 signaling during complete nitrogen starvation.

Anti-HA Western blots of protein extracts from wild-type and mutant cells expressing Sch9-3xHA from its native locus as they transition from (a) growth in SD+Gln to growth in SD+Pro, or (b) growth in SD complete medium to growth in SD-N medium. Quantification of Sch9 mobility data (n = 3) is shown on the right (see Supplementary Table 3 for p-values). Open circles and error bars show the average and standard deviation for each timepoint; filled circles show the data from individual experiments. The data were analyzed, and the graphs plotted, as described in Fig. 5.

Fig. 9. Model of multilayered TORC1 regulation across nitrogen limitation and starvation.

In glutamine, a high-quality nitrogen source, cells grow rapidly with only low-level transporter and metabolic reprogramming relative to SD complete medium. Upon a transition to nitrogen limitation or starvation, Ait1, Gcn2, and—to a lesser extent—SEAC cooperate to inactivate TORC1, causing growth inhibition and metabolic reprogramming. Over time, however, the outcomes diverge: (1) under nitrogen limitation (SD+Pro), cells enter a Low Nitrogen Adaptive (LoNA) state, maintaining metabolic reprogramming while partially reactivating TORC1 to support slow growth; whereas (2) under nitrogen starvation (SD-N), cells sustain strong TORC1 inhibition driven by SEAC together with Ait1 and Gcn2, leading to growth arrest and entry into quiescence. TORC1-dependent kinases that are activated or repressed in each condition are indicated based on the analysis in Supplementary Table 1 and Fig. 1. See text for further details. Figure created in BioRender. Padilla, C. (2025) Biorender.com. s79920z.