Snf1 regulation of Gcn4 abundance requires Gcn2 and Gcn20. A and B, cells expressing HA-tagged Gcn4 protein, in combination with either wild-type (+) or complete deletions (Δ) of the SNF1, GCN2 or GCN20 genes as shown, were grown in SC-Ura medium containing 2% glucose. Protein extracts were prepared and analyzed by Western blotting with antibodies against either the HA epitope or Sse1.

Model for Snf1 inhibition of GCN4 translation. Gcn2 kinase is stimulated when its HisRS tRNA synthetase-like domain is bound by uncharged tRNA. Stimulation by the HisRS domain is accentuated by the Gcn1-Gcn20 complex and attenuated by phosphorylation of Gcn2 at Ser-577 (6, 45). The TOR kinase pathway inhibits Gcn2 by promoting phosphorylation of Ser-577. Dephosphorylation of Ser-577 by the PP2A and/or Sit4 phosphatase occurs when TOR is inhibited by rapamycin (43). Snf1 may inhibit the activation of Gcn2 by either promoting phosphorylation of Ser-577 (directly or indirectly through the TOR pathway) or the inhibition of PP2A or Sit4 phosphatase. An alternative but not mutually exclusive model is that Snf1 inhibits Gcn2-dependent translation of Gcn4 by inhibiting the Gcn1-Gcn20 complex.

ARG1 expression in the absence of Snf1 requires Gcn4 activity. Total RNA was extracted from KY1200 (wild type), PY1012 (snf1Δ), PY1003 (gcn4Δ), or PY1008 (snf1Δ gcn4Δ) cells. Cells were grown in SC complete medium containing 2% glucose (Glu), and a portion of the culture was washed and transferred to SC complete medium containing 2% raffinose and 0.05% glucose (Raf) for 160 min. RNAs were detected by Northern blot analysis with probes for ARG1, GCN4, or SCR1 as shown. SCR1 RNA levels serve as a loading control.

Analog-sensitive allele of Snf1. A, halo assay of cells expressing wild-type Snf1 (bottom) or Snf1-I132G (top). Cells were spread on agar plates with sucrose as the carbon source. Sterile paper disks were spotted with either Me₂SO or inhibitor compounds dissolved in Me₂SO as indicated. B, structure of 2NM-PP1. C, growth curve of PY855 (Snf1) or PY856 (Snf1-I132G) cells grown in SC medium lacking uracil with 2% raffinose and 0.05% glucose as the carbon sources. Cells expressing either wild-type Snf1 (open symbols) or Snf1-I132G (filled symbols) were grown to an A₆₀₀ of 0.3, at which time Me₂SO (circles) or 2NM-PP1 dissolved in Me₂SO (squares) was added to a final concentration of 25 μm.

2NM-PP1 treatment of strains expressing Snf1-I132G reduces SUC2 transcription. Northern blot analysis of total yeast RNA (10 μg per lane) prepared from PY855 (Snf1) or PY856 (Snf1-I132G) cells is shown. Filters were probed sequentially with sequences from the SUC2 and ACT1 genes as shown. Cells were grown in 2% glucose and then shifted to medium containing 2% raffinose and 0.05% glucose for 2 h. Cultures were divided and treated with Me₂SO or 2% glucose as controls or with varying concentrations of 2NM-PP1 dissolved in Me₂SO for the indicated times.

Snf1 kinase activity inhibits expression of many Gcn4-dependent genes. Total RNA was prepared from cells grown in 2% glucose (G) or shifted to 2% raffinose and 0.05% glucose (R) for 160 min. FY1193 cells were transformed with pRS316 (snf1Δ), pSNF1-316 (SNF1), or pSNF1-I132G-316 (snf1-as). For the samples shown in lanes 4 and 5, cells were treated with 25 μm 2NM-PP1 dissolved in Me₂SO for 40 min prior to RNA extraction. RNA was detected by Northern blot analysis with the probes indicated to the right of each panel. Grouped data were obtained using successive hybridizations to the same filter. The data for ARG1 were all from the same scanned film, with the lanes rearranged for consistency with the other data presented here. SCR1 encodes the RNA subunit of the signal recognition particle and was used as an RNA loading control.

Microarray analysis of Snf1-I132G. A, scatter plot of mRNA abundance in cells expressing Snf1 or Snf1-I132G and treated with 2NM-PP1. FY1193 (snf1Δ) cells expressing either wild-type Snf1 (pSNF1-316) or Snf1-I132G (pSNF1-I132G-316) were transferred to SC-Ura medium containing 2% raffinose and 0.05% glucose for 2 h and incubated in the presence of 2NM-PP1 for 40 min. RNA was processed for microarray analysis (see “Experimental Procedures”). Mean log₂ values from two independent microarray data sets were plotted with wild-type Snf1 on the x axis and Snf1-I132G on the y axis. Genes differentially expressed in the absence of Snf1 kinase activity are shown above and below the lines indicating mean log₂ differences of 1.5. Genes whose expression is Gcn4-dependent (27) are shown in dark gray. B, scaled Venn diagram showing intersection of genes up-regulated in Snf1-I132G cells treated with inhibitor and genes down-regulated in the absence of Gcn4 (26). C, scaled Venn diagram showing intersection of genes up-regulated in Snf1-I132G cells treated with inhibitor and genes predicted to be direct targets of Gcn4 (27).

Snf1-mediated regulation of Gcn4 protein levels requires the small open reading frames in the GCN4 mRNA 5' leader region. A, wild-type SNF1 (PY1070, +) and snf1Δ (PY1064, Δ) cells expressing HA-tagged Gcn4 were grown in SC complete medium containing 2% glucose (Glu), and a portion of the culture was washed and transferred to SC complete medium containing 2% raffinose and 0.05% glucose (Raf) for 160 min. Protein extracts were prepared and analyzed by Western blotting with antibodies directed against HA or Sse1. B, wild-type SNF1 (PY1003) or snf1Δ (PY1008) cells were transformed with plasmids pPS128 (Gcn4-13myc, lanes 1 and 2) or pPS127 (Gcn4c-13myc, lanes 3 and 4). Cells were grown in SC-Ura medium containing 2% glucose. Protein extracts were prepared and analyzed by Western blotting with antibodies directed against the c-Myc epitope or Sse1. In an average of five experiments, normalized Gcn4c-13myc levels were only 1.14-fold higher in snf1Δ cells compared with SNF1 cells. C, wild-type SNF1 (PY1070) and snf1Δ (PY1064) cells were transformed with plasmids encoding β-galactosidase fused after amino acid 55 of the GCN4 open reading frame. The GCN4c-lacZ plasmid contains mutations that disrupt the small open reading frames in the GCN4 mRNA 5′ region. Extracts were prepared from cells grown in glucose medium and assayed for β-galactosidase activity. Error bars show mean ± 1 S.E. D, wild-type (WT) SNF1 (PY1094), snf1Δ (PY1095), gal83Δ (MSY522), sip1Δ (MSY528), sip2Δ (MSY520), gal83Δ sip1Δ (MSY552), gal83Δ sip2Δ (MSY543), sip1Δ sip2Δ (MSY544), gal83Δ sip1Δ sip2Δ (MSY557), and snf4Δ (MSY848) cells were transformed with the GCN4-lacZ plasmid. To control for the histidine prototrophy of the strains lacking the β subunits, SNF1 HIS3 and snf1Δ HIS3 strains were used in this analysis. Extracts were prepared and assayed as described in C. The source of the higher β-galactosidase levels in the absence of all three β subunits is unknown but could be due to an indirect effect of expressing three copies of HIS3, a Gcn4-regulated gene, in the absence of a functional Snf1 complex.