(A,B) The tap42-11 mutation alters the effects of rapamycin on phosphorylation of Ser 577 in GCN2 and Ser 51 in eIF2α at 30°C. Experiments analogous to those described in Figure 4A and B were carried out identically except that the strains were grown to early logarithmic phase at 25°C in SC medium lacking uracil or SC complete and transferred to the same medium prewarmed to 30°C for 30 min, after which they were treated with 200 ng/mL rapamycin for 20 min. (C,D) Effects of inactivating TAP42-regulated phosphatases on dephosphorylation of S577 and eIF2α phosphorylation. Transformants of strains BY4741 (WT), #3744 (sit4Δ), and CY1090 (pph21Δ pph22Δ) containing GCN2-FL on pDH101, were grown on SC medium lacking uracil to early logarithmic phase at 30°C and treated with 200 ng/mL rapamycin for 20 min, or were left untreated. Phosphorylation of Ser 577 in FL-GCN2 (C) or Ser 51 in eIF2α (D) was detected and quantified as described in Figure 1. Strain BY4741 transformed with pCB149 containing GCN2-S577A-FL (C) or gcn2Δ strain ESY6053 transformed with empty vector (D) was analyzed in parallel (lane 1).

(A) Phosphorylation of eIF2α on Ser 51 contributes to the inhibition of cell growth by rapamycin. Strains CY1100 (SUI2) and CY1101 (SUI2-S51A) were grown to saturation in liquid SC medium and serial dilutions were spotted to SC or SC plates containing 100 ng/mL rapamycin and incubated at 30°C for 2 and 4 d, respectively. (B) Phosphorylation of eIF2α on Ser 51 contributes to the inhibition of translation initiation by rapamycin. Strains CY1100 (SUI2) and CY1101 (SUI2-S51A) were grown in SC medium to early logarithmic phase and either treated with 200 ng/mL rapamycin for 2 h [(+) Rap.] or left untreated [(−) Rap.]. Fifty micrograms per milliliter cycloheximide was added 5 min before harvesting. WCEs were prepared and resolved on 5%–45% sucrose gradients. (P/M) Polysome-to-monosome ratio of the A₂₅₄ in all polysome fractions to that in the 80S peak.

Model depicting the TAP42/Ser 577-dependent pathway and a partially redundant uncharacterized pathway used by TOR to down-regulate GCN2 activity when nitrogen is plentiful. Phosphorylation of Ser 577 inhibits the GCN2 protein kinase (PK) domain by antagonizing tRNA binding. Treatment with rapamycin inhibits TOR, leading to dephosphorylation of Ser 577 by the TAP42-regulated phosphatases SIT4/PP2A. The ensuing activation of GCN2 and eIF2α phosphorylation down-regulates general translation initiation and induces GCN4 translation. The GCN4 thus produced activates amino acid biosynthetic genes and augments the GLN3-dependent activation of genes required for catabolism of poor nitrogen sources. Because rapamycin can partially induce eIF2α phosphorylation in tap42-11 cells at 25°C even though Ser 577 remains phosphorylated, we propose that a redundant pathway exists for TOR-dependent inactivation of GCN2 that is also inhibited by rapamycin. Starvation for amino acids can lead to activation of GCN2 through binding of uncharged tRNA to the HisRS domain even under conditions of TOR-dependent Ser 577 phosphorylation.

Effect of purine starvation and drug treatments on eIF2α phosphorylation on Ser 51 and GCN2 phosphorylation on Ser 577. (A) Transformants of gcn2Δ strain H2511 bearing centromeric plasmid pDH101, encoding Flag epitope-tagged GCN2 (FL-GCN2), were grown to early logarithmic phase in SD medium, and the cultures were split and cultured either untreated or treated with 50 μg/mL azaadenine (azA) for 20 min or 6 h. WCEs were prepared and phosphorylation of Ser 51 in eIF2α was detected by Western analysis with antibodies specific for eIF2α phosphorylated on Ser 51 (eIF2α-P) and compared with the total amount of the protein detected with antibodies against eIF2α. To detect phosphorylated Ser 577 in GCN2, we immunopurified FL-GCN2 from WCEs with anti-Flag M2 agarose affinity gel, resolved it by SDS-PAGE, and subjected it to Western analysis using antibodies specific for GCN2 phosphorylated on Ser 577 (Ser 577-P) or antibodies against total GCN2 (FL-GCN2). (B–D) Transformants described in A were grown in SC medium lacking uracil and treated with 0.0004% or 0.002% tunicamycin, 200 ng/mL rapamycin (Rap.), 0.07% MMS, or 50 mM HU for the indicated times, or were cultured without treatment to early logarithmic phase. Phosphorylation of Ser 51 in eIF2α (B) and Ser 577 in GCN2 (C,D) was measured as described in A. In C the first lane contained WCE from an isogenic transformant harboring empty plasmid pRS316. The odd-numbered lanes in D contained twice as much WCE as for the subsequent even-numbered lanes.

(A) Effect of rapamycin on phosphorylation of eIF2α is strongly dependent on dephosphorylation of Ser 577 and the tRNA binding activity of GCN2. Transformants of gcn2Δ strain H2511 bearing plasmid pDH101 containing GCN2-FL (WT), pCB149 containing GCN2-S577A-FL (S577A), pCB150 containing GCN2-m2-FL (m2), pCB151 containing GCN2-S577A-m2-FL (m2 S577A), or empty vector (gcn2Δ) were grown to early logarithmic phase in SC medium lacking uracil. The cultures were split in half and cultured either untreated (Rap.−) or treated with 200 ng/mL rapamycin for 20 min (Rap.+). WCEs were prepared, resolved by SDS-PAGE, and subjected to Western analysis using antibodies against total eIF2α or phosphospecific antibodies against eIF2α phosphorylated on Ser 51 (eIF2α-P). Western signals were quantified by video densitometry using NIH Image software (version 1.61), and the average ratios of eIF2α-P/eIF2α signals measured in three independent experiments are shown graphically below the relevant lanes with standard errors shown as error bars. The ratio in wild type was assigned a value of unity. (B) Induction of GCN4 expression by rapamycin is dependent on the uORFs. Transformants of wild-type strain BY4741 harboring CEN plasmids containing the GCN4–lacZ reporter with all four uORFs (p180) or lacking the upstream AUG codons of the uORFs (p227) were grown to early logarithmic phase in SC medium lacking uracil. The cultures were split in half and either cultured untreated (0 min) or treated with 200 ng/mL rapamycin for 30 or 120 min. Units of β-galactosidase activity (defined as nmole of o-nitrophenol β-D-galactopyranoside cleaved per minute per milligram of protein) were measured in WCEs from three or more independent transformants and the mean values and standard errors are depicted graphically. (C) Evidence that rapamycin activates GCN2 rather than inhibiting an eIF2α phosphatase. Transformants of GCN2 strain F113 bearing empty vector pRS316 (lanes 1,2), or transformants of gcn2Δ strain H2511 bearing p1246 containing HRI under the GAL promoter (lanes 3,4), or p1056 containing GCN2^c-E537K,E1522K (lane 5), or empty vector pRS316 (lane 6) were grown to early logarithmic phase in SC medium lacking uracil and containing raffinose as the carbon source. The cultures were split in half, and cultured either untreated or treated with 200 ng/mL rapamycin for 20 min. WCEs were prepared and analyzed as described for A.

The rapamycin-induced phosphorylation of Ser 51 in eIF2α and dephosphorylation of Ser 577 in GCN2 are both mediated by TOR proteins. (A) Overexpression of TOR2 from the GAL promoter or dominant TOR mutations that confer rapamycin resistance overcome the stimulation of eIF2α phosphorylation by rapamycin. Transformants of wild-type strain F113 with empty vector (lanes 1,2) or pJK5 expressing TOR2 under the GAL promoter (lanes 3,4) growing exponentially on SC medium lacking uracil were induced with galactose for 5 h and then treated with 200 ng/mL rapamycin for 20 min or were left untreated. (Lanes 5–11) Isogenic WT, TOR1-4, and TOR2-1 strains (MLY41, MLY90-1, and MLY152, respectively) were grown to early logarithmic phase on YPD medium and treated with rapamycin at 200 ng/mL for 20 min or left untreated. The gcn2Δ strain is H2511. Levels of eIF2α phosphorylation on Ser 51 in WCEs were measured as described in Figure 1A. (B) The TOR1-4 mutation abolishes dephosphorylation of Ser 577 in response to rapamycin. Transformants of strains MLY41 (WT) and MLY90-1 (TOR1-4) expressing FL-GCN2 from plasmid pDH101 (lanes 2–5) or MLY41 expressing GCN2-S577A-FL from pCB149 (GCN2-S577A) were grown in SC medium lacking uracil and treated with 200 ng/mL rapamycin for 20 min or were left untreated. Levels of FL-GCN2 phosphorylated on Ser 577 were measured as described in Figure 1A. Phosphorylation of Ser 577 in GCN2 was detected with antibodies that specifically recognize phosphorylated Ser 577, as it was described in the Figure 1 legend. (C) The tap42-11 mutation reduces induction of GCN4 expression by rapamycin. Transformants of TAP42 strain CY1077 or tap42-11 strain CY1078 harboring p180 containing the GCN4–lacZ reporter with all four uORFs were grown for the indicated times in the presence of rapamycin and levels of β-galactosidase activity in the WCEs were measured, all as described in Figure 2B.

The tap42-11 mutation diminishes the effects of rapamycin in promoting dephosphorylation of Ser 577 in GCN2 and increased phosphorylation of Ser 51 in eIF2α at 25°C. (A) Transformants of strains CY1077 (TAP42) and CY1078 (tap42-11) containing GCN2-FL on pDH101 (lanes 2–5), or CY1077 transformed with pCB149 containing GCN2-S577A-FL (lane 1), were grown on SC medium lacking uracil to early logarithmic phase at 25°C and either treated with 200 ng/mL rapamycin for 2 h or left untreated. WCE extracts were prepared and analyzed for phosphorylation of Ser 577 in FL-GCN2 as described in Figure 1. (B) Strains CY1077 (TAP42) and CY1078 (tap42-11) were cultured in SC medium and analyzed for eIF2α phosphorylation as described in Figure 1. The gcn2Δ strain ESY6053 was analyzed in parallel (lane 1). (C,D) Multiple experiments analogous to those described in A and B were carried out identically except that the rapamycin treatment was for 30 min or 4 h. The eIF2α-P/eIF2α and S577-P/FL-GCN2 ratios were calculated as described in Figure 2A and plotted against the hours of rapamycin treatment.