Glucose availability regulates phosphorylation of Gln3. (A) Glucose starvation decreases Gln3 gel mobility. Exponentially growing cells in SC medium (OD₆₀₀ = 0.4) were switched to SC medium, SC medium plus rapamycin (SXC+RAP), SC medium minus nitrogen (SC-Nitrogen), or SC medium minus glucose (SC-glucose) for 0, 10, and 30 min. Gln3 gel mobility was detected by Western blotting. (B) Electrophoretic decrease of Gln3 during glucose limitation is due to phosphorylation. Exponential-phase yeast cells (OD₆₀₀ = 0.4) in SC medium (lane 1) were switched to SC medium minus glucose for 30 min (lane 2). Lysates of cells under glucose starvation were incubated with a buffer for CIP (lanes 3 and 4) or CIP plus Na₄P₂O₇ (lane 5). Gln3 electrophoretic mobility was detected by Western blotting with MAb 9E10. (C) Phosphorylation of Gln3 during glucose limitation is reversible. Yeast cells cultured in SC medium minus glucose (SC-Glc) were switched to SC medium for 10 and 30 min. Gln3 gel mobility was detected by Western blotting. (D) Glucose limitation causes phosphorylation of Gln3. Exponential cells in SC medium were switched to SC medium plus glucose or SC medium containing one of the following reagents as the sole carbon source for 30 min: ethanol, raffinose, or galactose. Gln3 gel mobility was detected by Western blotting.

The role of glucose sensors in the in vivo phosphorylation of Gln3. (A) Mutations in the Snf1 pathway confer differential rapamycin sensitivity. The wild-type (WT) and mutant yeast strains were streaked onto YPD and YPD plus rapamycin (YPD+RAP) (25 nM) plates and incubated at 30°C for 2 (YPD) and 4 (YPD plus rapamycin) days, respectively. (B) Snf1 is required for Gln3 phosphorylation during glucose starvation, but not during nitrogen starvation. Exponentially growing wild-type and snf1Δ cells expressing Gln3-MYC₉ in SC medium (OD₆₀₀ = 0.4) were switched to SC medium or SC medium minus glucose or nitrogen for 30 min. (C) Recombinant Snf1 restores the deficiency of Gln3 phosphorylation during glucose limitation in the snf1Δ strain. The snf1Δ strains carrying a plasmid expressing recombinant Snf1 or a vector control were examined for Gln3-MYC₉ phosphorylation at different times after glucose limitation. (Upper panel) Western blot for Gln3-MYC₉. (Lower panel) Western blot for Snf1. (D) Rgt2 and Snf3 are not required for Gln3 phosphorylation during glucose limitation. Exponentially growing wild-type and mutant cells expressing Gln3-MYC₉ in SC medium (OD₆₀₀ = 0.4) were switched to SC medium or SC medium minus nitrogen or glucose for 30 min. Gln3 phosphorylation was examined by Western blotting.

Snf1 is required for Gln3 nuclear accumulation and induction of NCR genes during glucose limitation. (A) Early-log-phase (OD₆₀₀ = 0.2) wild-type (WT) or snf1Δ yeast cells expressing Gln3-MYC₉ in SC medium were switched to SC medium, SC medium minus glucose (−glucose), or SC medium minus nitrogen (−Nitrogen) for 30 min. Gln3 localization was detected by indirect IF with the 9E10 MAb. DAPI, 4",6-diamidino-2-phenylindole. (B) Snf1 is required for expression of NCR genes during glucose limitation. Exponential wild-type or mutant yeast cells (OD₆₀₀ = 0.4) in SC medium were shifted to SC medium minus glucose. Aliquots of culture were withdrawn at different times (as shown) and analyzed for expression of selected NCR genes by Northern blotting.

Glucose availability regulates subcellular localization of Gln3 and Gln3-dependent NCR genes. (A) Glucose availability regulates subcellular localization of Gln3. Early-log-phase cells (OD₆₀₀ = 0.2) expressing Gln3-MYC₉ in SC medium containing 2% glucose were switched to SC medium, SC medium minus glucose, or SC medium minus nitrogen (SC medium minus ammonium sulfate and amino acids) for 30 min. Gln3-MYC₉ localization was then analyzed by indirect IF staining with MAb 9E10. Nuclear DNA was stained with 4",6-diamidino-2-phenylindole. The cell staining images were captured with a SPOT digital camera. (B) Glucose availability regulates Gln3-dependent NCR genes. Exponentially growing wild-type and mutant yeast cells (OD₆₀₀ = 0.4) in SC medium were switched to SC medium minus glucose. Samples were withdrawn at different times and analyzed for expression of GAP1, GDH2, PUT1, and ACT1 by Northern blot.

TOR is not required for phosphorylation of Gln3 during glucose limitation. Exponentially growing yeast cells in SC medium (OD₆₀₀ = 0.4) were treated with rapamycin for 30 min to generate completely dephosphorylated Gln3. These cells were then switched to SC medium with or without glucose in the presence of rapamycin for 10 and 30 min. Shown is the gel mobility of Gln3 as detected by Western blotting.

Snf1 interacts with Gln3. (A) Snf1 interacts with Gln3 in a yeast two-hybrid assay. Gal4 DNA binding domain (BD)-Snf1 specifically interacts with Gal4 activation domain (AD)-Gln3, but not with AD alone. The two-hybrid reporter strain (AH109) carrying different BD and AD fusion plasmids was assayed for growth on an adenine dropout plate or a histidine dropout plate containing 3-AT (2 mM). LgT, large T antigen. (B) Snf1 binds to GST-Gln3. Extracts of wild-type (WT) and snf1Δ yeast strains were assayed for their ability to bind to immobilized, bacterially produced GST or GST-Gln3. The bound materials were detected by Western blotting with an anti-polyhistidine antibody that recognizes Snf1. (C) Snf1 phosphorylates Gln3 in vitro. HA-Snf1 was immunoprecipitated and assayed for its ability to phosphorylate bacterial recombinant GST-Gln3 in the presence of [γ-³²P]ATP.

Model for regulation of Gln3 by both nitrogen and glucose. TOR and Snf1 independently mediate nitrogen and glucose signaling pathways, respectively, to regulate Gln3 nuclear localization and specificity for its target genes. Our results suggest that phosphorylation controls the subcellular localization of Gln3. TOR-dependent phosphorylation appears to keep Gln3 in the cytoplasm, while Snf1-dependent phosphorylation leads to nuclear accumulation of Gln3 by promoting nuclear import and/or inhibiting nuclear export.