Sterol binding regulates Kes1 activity. (A) WT yeast (CTY182) engineered for Dox-repressible expression of the indicated Kes1 derivatives were spotted in 10-fold dilutions series on uracil-free media +/− Dox (10μg/ml) and incubated at 30°C. (B) Lysates were prepared from the strains in (A), normalized for total protein (10μg loaded), and Kes1 and GAPDH were visualized by immunoblotting. (C) WT yeast expressing Kes1-GFP or kes1^Y97F-GFP were cultured to mid-logarithmic growth phase in uracil-free medium at 30°C. GFP profiles are shown. (D) WT yeast expressing kes1^Y97F-GFP were pulse-labeled with FM4-64 (10μM) for 10 min and poisoned with a 10 mM NaN₃/NaF cocktail. FM4-64, GFP and merge profiles are shown with corresponding DIC image (bar = 5μm). (E) WT yeast expressing Kes1-GFP were challenged with miconazole for 4hrs. GFP profiles are shown. (F) pik1-101^ts cells expressing kes1^Y97F-GFP were cultured at 25°C, split and one culture maintained at 25°C while the partner was shifted to 37°C for 1hr prior to imaging. (G) Profiles of GFP-tagged versions of indicated Kes1 derivatives are shown with corresponding DIC images (bar = 5μm). (H) WT yeast engineered for Dox-repressible expression of indicated Kes1 derivatives (or mock Ura3 control) were spotted in 10-fold dilutions series on selective media with Dox (10μg/ml) as variable. Also see .

Kes1 and PtdIns-4-P homeostasis. (A) WT and pik1-101^ts cells expressing GOLPH3-GFP were cultured in uracil-free medium at 25°C and shifted to 37°C for 60 min prior to imaging. Corresponding DIC images are shown at bottom (bar = 5μm). (B) WT yeast (CTY8-11Cα) expressing GOLPH3-GFP and indicated Kes1 derivatives were incubated +/− Dox (10μg/ml) for 18 hours. GOLPH3-GFP (top panel) and DIC images (bottom panel) profiles are shown (bar = 5μm). (C) WT yeast (CTY182) engineered for Dox-repressible expression of Kes1 derivatives were radiolabeled to steady-state at 30°C with 20 μCi/ml [³H]-Ins +/− Dox as indicated. Fractional incorporation of [³H]-Ins into the deacylated PtdIns-3-P, PtdIns-4-P, and PtdIns(4,5)P₂ species is indicated (n = 4). Error bars represent standard deviation. (D) WT yeast (CTY182) expressing the Kes1 derivatives were cultured +/− Dox. Cells were pulse-radiolabeled with [³⁵S]-amino acids (15 min). After 30 min of chase, immunoprecipitated CPY species were analyzed by SDS-PAGE and autoradiography. p2 CPY and mCPY are indicated. (E) WT yeast (CTY182) expressing Kes1 species (−Dox) and corresponding non-expressing controls (+Dox), were examined for GFP-Snc1 distribution. DIC images are shown at bottom (bar = 5μm). Also see .

Kes1 and amino acid homeostasis. (A) WT yeast cells (CTY182) producing Kes1 derivatives (−Dox) and corresponding non-expressing controls (+Dox; 10μg/ml) were analyzed by EM. Representative images are shown (bar = 1μm). Intra-vacuolar vesicular profiles (diameter ~ 350nm) are highlighted by arrows. (B) The ALP-expressing PHY2433 strain (TN124; ) was induced (−Dox), or not (+Dox; 10μg/ml), for Kes1 or kes1^Y97F expression. Cultures were maintained at an OD_600nm < 0.2 throughout, harvested and assayed for ALP activity. Error bars represent standard deviation. (C) PCA scores plot distinguishing the ¹H-NMR metabolic profiles of WT yeast (CTY182) expressing Kes1 or kes1^Y97F (− Dox), or not (+Dox). (D) Targeted PCA of individual metabolites was performed for each condition. Reductions in Arg, Asn, Asp, Glu, Gln, Thr, and Trp pools were the major contribution to the variance between conditions of excess Kes1/kes1^Y97F and mock controls. (E) WT yeast (CTY182) transformed with YCp(URA3), YCp(P_DOX::KES1) or YCp(P_DOX::kes1^Y97F) were spotted in 10-fold dilution series on media with Dox (10μg/ml) and amino acids (NEQR, 0.2% w/v). (F) atg13Δ yeast co-transformed with YEp(P_CUP1::HA-ATG13) and either YCp(P_DOX::KES1) or YCp(P_DOX::kes1^Y97F) were cultured in media containing 10μg/ml Dox. KES1 or kes1^Y97F expression was induced for 7 hours (−Dox), or not (+Dox; 10μg/ml), and cells were challenged with CuSO₄ (100μM for 1hr) to induce HA-Atg13 expression. HA-Atg13 species were visualized by immunoblotting with anti-HA antibodies. Also see .

Kes1 attenuates Gcn4-dependent activation of the general amino acid control. (A) Lysates were prepared from paired cell cultures induced for Kes1 or kes1^Y97F expression (−Dox), or not (+Dox; 10μg/ml). Phospho-eIF2α, Kes1 and eIF2α were visualized by immunoblotting. (B) Total RNA was prepared from cells (CTY8-11Cα) induced for Kes1 or kes1^Y97F expression (−Dox), or not (+Dox) and challenged with 100mM 3-AT for 2 hours to induce the GAAC. HIS4, ARG1 and ACT1 expression was surveyed by RT-PCR. Error bars represent standard deviation. (C) Gcn4, Kes1 and GAPDH were analyzed by immunoblotting of lysates prepared from WT cells (CTY8-11Cα) expressing Kes1 or kes1^Y97F (−Dox) or not (+Dox). Cells were challenged with 100mM 3-AT for 2 hours to induce the GAAC. (D) Total RNA fractions were isolated from WT cells harboring YCp(P_DOX::KES1) and co-transformed with YCp (TRP1) or YCp (GCN4^C) cultured +/− Dox. HIS4 and ARG1 gene expression was surveyed by RT-PCR, and normalized to ACT1 expression. Undiluted product and a 4-fold dilution of product was analyzed. Error bars represent standard deviation. (E) The experiment is as in (D) with the modification that an NEQR cocktail (0.2% w/v) was added to a parallel culture induced for Kes1 expression. HIS4, ARG1 and ACT1 expression were scored by RT-PCR. Error bars represent standard deviation. (F) RT-PCR of GAAC target genes ARG1 and HIS4, as well as ACT1 control from total RNA fractions prepared from WT, sec14-1^ts tlg2Δ, sec14-1^ts tlg2Δ kes1Δ or sec14-1^ts tlg2Δ cki1Δ cells shifted to 37°C for 2hrs to impose the strong sec14-1^ts tlg2Δ-associated TGN/endosomal trafficking block. Error bars represent standard deviation. Also see .

Sphingolipids inhibit the GAAC. (A) Quantitative lipidomics of DHC, PHC, DHS, PHS, DHS-1-P and PHS-1-P in yeast (CTY182) as a function of Kes1 or Kes1^Y97F expression. Note the log₁₀ scale for the y-axis. (B) WT strain (CTY8-11Cα) and a kes1Δ derivative were spotted in a 10-fold dilution series on synthetic complete (SC) media and SC media with 7.5μM PHS, with 10mM 3-AT, or both. (C) Total RNA fractions were isolated from WT yeast either mock treated or grown in the presence of 7.5μM PHS for 2 hours. Cells were either mock-treated or challenged with 100mM 3-AT for 2 hours to induce the GAAC. HIS4, ARG1 and ACT1 expression were monitored by RT-PCR. Undiluted product and a 4-fold dilution of product were analyzed. Error bars represent standard deviation. (D) WT yeast (CTY8-11Cα) expressing Ura3 or Gcn4^c were spotted in a 10- fold dilution series on SC media and SC media with 7.5μM PHS, with 10mM 3-AT, or both. (E) Total RNA fractions were prepared from WT yeast expressing Ura3 or Gcn4^c +/− 15μM PHS for 2 hrs. HIS4 and ARG1 expression was surveyed by RT-PCR and normalized to ACT1 expression. Undiluted product and a 4-fold dilution of product were analyzed. Error bars represent standard deviation. Also see .

Gcn4 binding to target genes and Mediator. (A) Cells were treated with PHS (15μM) for 2 hrs prior challenge with 3-AT (10mM) for 30 min, and perfused with formaldehyde. Sonicated chromatin was precipitated with antibodies against Gcn4 (A) or Rpb3 (B). DNA extracted from immunoprecipitates (ChIP) or starting chromatin (input) was PCR-amplified in the presence of [³²P]-dATP with primers for ARG1 UAS (A), TATA element, or sequences from the 5′ or 3′ end of the ARG1 coding sequences (B), and with primers for an intergenic chromosome V sequence (nonspecific control). PCR products were quantified by phosphorimaging. Ratios of ARG1 ChIP-to-input signals were normalized against chromosome V control sequence ratios to yield occupancy values. Averages and standard errors from 2 PCR amplifications for each of 2 independent immunoprecipitates from 2 independent cultures are plotted. Error bars represent standard deviation. (C) Isogenic WT (CTY1740) and srb10Δ yeast harboring YCp vectors for Dox-repressible expression of Kes1 were spotted in 10-fold dilutions series on media +/− Dox (10μg/ml) and incubated at 30°C. (D) Isogenic WT (CTY1740) and srb10Δ yeast were spotted in a 10-fold dilution series on media with 7.5μM PHS, with 10mM 3-AT, or both 7.5μM PHS and 10mM 3-AT. (E) Isogenic WT (CTY1740) or srb10Δ yeast were either mock treated, or challenged with 15μM PHS for 2 hrs. The cultures were challenged with 100mM 3-AT for 2 hrs to induce the GAAC. HIS4, ARG1 and ACT1 expression was monitored by RT-PCR. Undiluted product and a 4-fold dilution of product was analyzed. Error bars represent standard deviation.

Integration of TGN/endosomal trafficking flux to transcriptional competence of the nitrogen response. (A) Membrane trafficking through a signaling TGN/endosomal compartment is defined by Sec14-regulated production of PtdIns-4-P countered by Kes1-mediated PtdIns-4-P sequestration. Degree of sequestration is set by PtdIns-4-P-mediated recruitment of Kes1 to TGN/endosomal membranes, and released by binding of the membrane-bound Kes1 to active sterol. (B) Kes1-mediated trafficking defects reduce efficiency of amino acid and NH₄⁺ permease delivery to the plasma membrane. The incipient nitrogen stress results in reduced TOR activity and engagement of the early stages of the GAAC (phosphorylation of eIF2α). Efficiency of the terminal stages of the GAAC is determined by potency of Gcn4 antagonism levied by activity of the large SRB Mediator complex. (C) Potency of a Kes1-mediated trafficking arrest/delay sets the amplitude of TGN/endosomal production of SL available for nuclear signaling. SL* denotes a sphingolipid with signaling power (e.g. ceramide) produced in TGN/endosomal compartments. The potency of SL* signaling is directly proportional to Kes1 activity, and inhibits transcription initiation/elongation of genes loaded with Gcn4 and RNA polymerase II via action of the CDK8 module of the large SRB Mediator complex. SL* signaling foils the GAAC and induces cells to enter a metabolically coherent quiescence with properties of post-mitotic cell physiology.