The TOR-controlled transcription activators GLN3, RTG1, and RTG3 are regulated in response to intracellular levels of glutamine - PubMed (original) (raw)
The TOR-controlled transcription activators GLN3, RTG1, and RTG3 are regulated in response to intracellular levels of glutamine
José L Crespo et al. Proc Natl Acad Sci U S A. 2002.
Abstract
The essential, rapamycin-sensitive TOR kinases regulate a diverse set of cell growth-related readouts in response to nutrients. Thus, the yeast TOR proteins function as nutrient sensors, in particular as sensors of nitrogen and possibly carbon. However, the nutrient metabolite(s) that acts upstream of TOR is unknown. We investigated the role of glutamine, a preferred nitrogen source and a key intermediate in yeast nitrogen metabolism, as a possible regulator of TOR. We show that the glutamine synthetase inhibitor L-methionine sulfoximine (MSX) specifically provokes glutamine depletion in yeast cells. MSX-induced glutamine starvation caused nuclear localization and activation of the TOR-inhibited transcription factors GLN3, RTG1, and RTG3, all of which mediate glutamine synthesis. The MSX-induced nuclear localization of GLN3 required the TOR-controlled, type 2A-related phosphatase SIT4. Other TOR-controlled transcription factors, GAT1/NIL1, MSN2, MSN4, and an unknown factor involved in the expression of ribosomal protein genes, were not affected by glutamine starvation. These findings suggest that the TOR pathway senses glutamine. Furthermore, as glutamine starvation affects only a subset of TOR-controlled transcription factors, TOR appears to discriminate between different nutrient conditions to elicit a response appropriate to a given condition.
Figures
Figure 1
(A) Schematic drawing of the TCA cycle and metabolic reactions for the synthesis of glutamine. α-KG, α-ketoglutarate. (B) Effect of MSX on the size of the intracellular pools of glutamine and glutamate. Wild-type (JK9–3da) cells were grown in SD and treated with MSX. Time refers to MSX treatment. MSX was added at 0 min. A 100% glutamine pool corresponds to 13.1 μmol/g of protein; a 100% glutamate pool corresponds to 42.4 μmol/g of protein. Open triangles refer to the intracellular accumulation of MSX. A 100% MSX pool corresponds to 19.9 μmol/g of protein. (C) Effect of the GS inhibitor MSX on cell growth. Wild-type (JK9–3da) cells were grown in SD and treated with MSX (2 mM) (▵) or simultaneously with MSX and glutamine (5 mM) (▴). Control refers to nontreated cells (●). The arrow indicates time of treatment.
Figure 2
Glutamine starvation specifically activates the transcription factor GLN3 but not GAT1. (A) Localization of GLN3-myc (GLN3) in a wild-type (wt) (TB123) and gln1 (JC33–6c) and sit4 (TB136–2a) mutant cells. Wild-type cells were grown in SD and treated either with rapamycin (+ rap) or MSX (+ MSX) for 20 min. Wild-type cells also were treated with MSX for 20 min and then with glutamine (5 mM final concentration) for 5 min (+ MSX + Gln). Untreated wild-type cells were used as a control. gln1 mutant cells were grown in SD supplemented with 0.3% glutamine and shifted to glutamine-free medium for 20 min. sit4 mutant cells were treated with MSX for 20 min. All strains were grown in SD at 30°C. Cells and DNA were visualized by Nomarski optics and 4′,6-diamidino-2-phenylindole staining (DNA). (B) GLN3 is dephosphorylated in glutamine-starved cells. Wild-type cells (TB123) were grown in SD and treated with either rapamycin or MSX for 25 min. GLN3-myc was detected by immunoblotting. (C) Reverse transcriptase–PCR analysis of total RNA from wild-type (JK9–3da) and gln3 (TB103–1d) mutant cells with oligonucleotide primers specific to the actin gene (ACT1) and two nitrogen-regulated genes, GLN1 and MEP2. Cells were grown in SD to an OD600 of 0.5 and treated with MSX for either 20 or 40 min. Nontreated cells (time 0) were used as a control. (D) GAT1-HA (GAT1) was visualized by indirect immunofluorescence in wild-type (TB106–2a) cells untreated (Control) or treated with rapamycin (+ rap) or MSX (+ MSX) for 20 min. (E) Growth of wild-type (JK9–3da), gln3 (TB103–1d), gat1 (TB102–1a), and gln3 gat1 (TB105–3b) mutant cells in SD or SD supplemented with a sublethal concentration (1 mM) of MSX. Plates were incubated at 30°C for 3 days.
Figure 3
Glutamine starvation activates the transcription factors RTG1 and RTG3. (A) RTG1 localized in the nucleus in glutamine-depleted cells. Wild-type (JC37–1a) cells expressing a functional RTG1-green fluorescent protein (GFP) fusion protein were grown in SD containing 5 mM glutamate and treated either with rapamycin (+ rap) for 15 min or MSX (+ MSX) for 30 min. (B) Induction of the RTG-target gene CIT2 by rapamycin and MSX. Wild-type (K699) cells grown in glutamate-containing SD were treated with rapamycin and/or MSX for 30 min in the presence or the absence of glutamine. Cells were then harvested and total RNA was prepared and analyzed by Northern blotting, blotting for the specified mRNAs (Top). (C) RTG genes mediate the induction of CIT2 in MSX-treated cells. Wild-type (K699) cells and rtg1 (EY0733), rtg2 (EY0734), or rtg3 (EY0735) mutant cells were grown in glutamate-containing SD and treated with MSX for 30 min. Cells were then harvested and total RNA was isolated. Expression of CIT2 and ACT1 was determined by Northern blotting as described in Materials and Methods.
Figure 4
TOR regulates a specific subset of proteins in response to glutamine. (Left) In the presence of nutrients, TOR keeps the transcription factors GLN3, GAT1, RTG1/3, and MSN2/4 inactive. (Right) Glutamine is the specific signal that regulates GLN3, RTG1, and RTG3, whereas GAT1, MSN2, and MSN4 are regulated by other metabolites. The question mark refers to an unknown transcription factor that regulates r-protein genes.
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