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.