Rapamycin induces and represses gene expression. The wild-type yeast strain MLY41 was grown to early exponential phase, and treated with or without 0.2 μg/ml rapamycin or 10 μg/ml cycloheximide for 15, 30, 60, and 120 min, as indicated. RNA was prepared and analyzed by Northern blot with radioactive probes that hybridize to the genes indicated at left. Hybridization to the actin gene (ACT1) served as a loading control.

Mutations affecting nitrogen-source utilization alter rapamycin toxicity. (A) Isogenic wild-type (MLY41) and Δgln3 (MLY139), Δure2 (MLY140a), Δnpr1 (MLY54a), npi1 (MLY141a), and fpr1 (MLY88) mutant strains were grown on YPD medium containing 0, 10, or 50 ng/ml rapamycin and incubated for 3 days at 30°C. (B) Isogenic wild-type (MLY41) and Δure2 (MLY140a), TOR2-1 (MLY152α), TOR2-1 Δure2 (MLY158α), TOR1-4 (MLY90-1), TOR1-4 Δure2 (MLY148α), fpr1 (MLY88), and fpr1 Δure2 (MLY146a) mutant strains were grown on YPD medium containing 0 or 100 ng/ml rapamycin and incubated for 3 days at 30°C.

Tor and nutrients regulate the phosphorylation state of Ure2. (A) The isogenic wild-type (WT, strain MLY41) and Δfpr1 mutant strain (strain MLY88) were grown to early exponential phase (OD₆₀₀ = 0.4) in YPD medium and treated with 0.2 μg/ml rapamycin for 0, 15, and 30 min. Alternatively, the isogenic wild-type strain MLY41 was grown as above, washed with SLAD ammonium limiting medium, and then incubated in SLAD medium for 15 and 30 min. Total cell extracts were prepared and 100 μg of protein analyzed by SDS-PAGE (12.5%) and Western blot with Ure2 antibodies. Cell extract from a Δure2 mutant strain (strain MLY140a) was included as a control for antisera specificity. (Arrows) The migration position of phosphorylated isoforms of Ure2; (*) a protein that crossreacts nonspecifically with the anti-Ure2 antisera. (B) Protein cell extract from wild-type strain MLY41 was incubated with 4 units of alkaline phosphatase for 30 min at 37°C. Protein extracts from rapamycin treated or untreated wild-type strain (MLY41) or the Δfpr1 mutant strain (MLY88), as indicated, were mock incubated and loaded in lanes 1, 3, 4, and 5 to compare the mobility of phosphorylated and dephosphorylated Ure2. Note that cell extracts in A were analyzed in 12.5% SDS–polyacrylamide gels; the extracts in B were fractionated in 10% SDS-PAGE for a longer period of time, which could explain the greater separation of phosphorylated and dephosphorylated Ure2 observed in B.

The TOR signaling cascade regulates gene expression in response to nutrients. The Tor proteins are activated by nutrients, and regulate the expression of genes involved in the utilization of nitrogen sources via Ure2 and Gln3, ribosome biogenesis by RNA polymerases, and amino acid permease stability.