General Amino Acid Control and 14-3-3 Proteins Bmh1/2 Are Required for Nitrogen Catabolite Repression-Sensitive Regulation of Gln3 and Gat1 Localization

Abstract

Nitrogen catabolite repression (NCR), the ability of Saccharomyces cerevisiae to use good nitrogen sources in preference to poor ones, derives from nitrogen-responsive regulation of the GATA family transcription activators Gln3 and Gat1 In nitrogen-replete conditions, the GATA factors are cytoplasmic and NCR-sensitive transcription minimal. When only poor nitrogen sources are available, Gln3 is nuclear, dramatically increasing GATA factor-mediated transcription. This regulation was originally attributed to mechanistic Tor protein kinase complex 1 (mTorC1)-mediated control of Gln3 However, we recently showed that two regulatory systems act cumulatively to maintain cytoplasmic Gln3 sequestration, only one of which is mTorC1. Present experiments demonstrate that the other previously elusive component is uncharged transfer RNA-activated, Gcn2 protein kinase-mediated general amino acid control (GAAC). Gcn2 and Gcn4 are required for NCR-sensitive nuclear Gln3-Myc¹³ localization, and from epistasis experiments Gcn2 appears to function upstream of Ure2 Bmh1/2 are also required for nuclear Gln3-Myc¹³ localization and appear to function downstream of Ure2 Overall, Gln3 phosphorylation levels decrease upon loss of Gcn2, Gcn4, or Bmh1/2 Our results add a new dimension to nitrogen-responsive GATA-factor regulation and demonstrate the cumulative participation of the mTorC1 and GAAC pathways, which respond oppositely to nitrogen availability, in the nitrogen-responsive control of catabolic gene expression in yeast.

Keywords: Bmh1/2; Gat1; Gcn2; Gln3; nitrogen catabolite repression.

Figure 1

Gcn2 is required for nuclear Gln3-Myc¹³ localization in response to growth with nitrogen sources whose use requires NCR-sensitive transcription. Gln3-Myc¹³ localization was measured in (A) wild-type (BY4742) and (B) gcn2Δ cells cultured in complex YPD or YNB-minimal medium with glutamine (Gln), ammonia (Am.), or proline (Pro) as sole nitrogen source. Rapamycin (+Rap) or methionine sulfoximine (+Msx) were added where indicated to glutamine- or ammonia-grown cells, respectively. Intracellular Gln3-Myc¹³ was visualized and scored as cytoplasmic (red bars), nuclear-cytoplasmic (yellow bars), or nuclear (green bars) as described in Materials and Methods. Nucl.-Cyto., nuclear-cytoplasmic.

Figure 2

Gcn2 requirement for nuclear Gln3-Myc¹³ localization in cells provided with poor, derepressive nitrogen sources. Gln3-Myc¹³ localization was measured in (A) wild-type (BY4742) and (B) gcn2Δ cells. Nitrogen sources ranged from derepressive to repressive: allantoin (All), γ-amino-butyrate (GABA), ammonia (Am.), serine (Ser), glutamine (Gln), or asparagine (Asn) were provided (final concentration of 0.1%) as sole nitrogen source in YNB-minimal media. The experimental format and data presentation were as in Figure 1. Nucl.-Cyto., nuclear-cytoplasmic.

Figure 3

Gcn2 is required for NCR-sensitive GDH2 gene expression when cells are cultured with derepressive nitrogen sources. GDH2 expression was measured in wild-type (BY4742) and gcn2Δ cells using qRT-PCR provided with glutamine (Gln), proline (Pro), or allantoin (All) as sole nitrogen source. Where indicated, glutamine-grown cells were treated with rapamycin. Total RNA was collected and analyzed as described in Materials and Methods. +Rap, addition of rapamycin.

Figure 4

The effects of Gcn2 abolishment on Gln3-Myc¹³ localization in response to short- and long-term nitrogen starvation. (A) Wild-type (BY4742) and (B) gcn2Δ cells were grown to A_{600 nm} = 0.5 in YNB-glutamine medium. They were then transferred (by filtration) to nitrogen-free YNB medium and sampled as indicated for 6 hr. At that time glutamine (0.1%) was added to the culture and sampling continued for 30 min. Gln3-Myc¹³ was visualized and scored as described in Materials and Methods. Nucl.-Cyto., nuclear-cytoplasmic.

Figure 5

The effects of Gcn2 abolishment on budding and Gln3-Myc¹³ localization in response to long-term nitrogen starvation. (A) Wild-type (BY4742) and (B) gcn2Δ cells were grown to A_{600 nm} = 0.5 in YNB-glutamine medium. They were then transferred (by filtration) to nitrogen-free YNB medium and sampled as indicated for 10 hr. Gln3-Myc¹³ was visualized and scored as described in Materials and Methods. The extent of cell budding (C) was scored in 200 or more cells per determination. Nucl.-Cyto., nuclear-cytoplasmic.

Figure 6

ure2Δ is epistatic to gcn2Δ. Wild type (BY4742), gcn2Δ, ure2Δ (RR259), and gcn2Δure2Δ (JT11) mutants were cultured in YNB-glutamine (Gln), -asparagine (Asn) or -proline (Pro) medium. (A) Intracellular Gln3-Myc¹³ localization was followed in the various mutants grown in asparagine or proline medium as described in Figure 1. (B) Total RNA was harvested as described in Materials and Methods and subjected to qRT-PCR analysis using a GDH2 probe as described in Figure 3. Nucl.-Cyto., nuclear-cytoplasmic.

Figure 7

Gcn2 and mTorC1 are coregulators of intracellular Gln3-Myc¹³ regulation. Wild type (BY4742) and gcn2Δ strains were transformed with wild type (pRR536) and gln3 substitution mutant plasmids pRR680, pRR850, pRR1045, and pRR1194. The transformants were then grown in YNB-proline medium and Gln3-Myc¹³ intracellular localization assayed. The format and presentation of the experimental data were as described in Figure 1. Nucl.-Cyto., nuclear-cytoplasmic; W.T., wild type.

Figure 8

(A) Gcn4 is required for nuclear Gln3-Myc¹³ localization in response to growth with nitrogen sources whose use requires NCR-sensitive transcription. Gln3-Myc¹³ localization was measured in wild-type (BY4742), gcn2Δ, and gcn4Δ cells cultured in YNB-minimal medium provided with allantoin (All), urea, or proline (Pro) as nitrogen source. The format and presentation of the experimental data were as described in Figure 1. (B and C) Effects of Gcn4 abolishment on Gln3-Myc¹³ localization and cell budding (D) during nitrogen starvation. The format of the experiment and presentation of the data are as described in Figure 5. Nucl.-Cyto., nuclear-cytoplasmic.

Figure 9

Gln3-Myc¹³ phosphorylation levels decrease when Gcn2 (Panels A-C) or Gcn4 (Panels D-F) is abolished. Cell-free extracts were prepared from wild-type (BY4742), gcn2Δ, or gcn4Δ cultures grown in YNB-ammonia (Am.), -proline (Pro), or -glutamine (Gln). Rapamycin (+Rap) or Msx (+Msx) were added as indicated. Proteins in the extracts were then resolved using SDS-PAGE and Western blots prepared and visualized as described in Materials and Methods. W.T., wild type.

Figure 10

Gcn2 is required for nuclear Gat1-Myc¹³ localization in response to growth with nitrogen sources whose use requires NCR-sensitive transcription. Cells were cultured in YNB-glutamine (Gln), -ammonia (Am.), or -proline (Pro) medium. Rapamycin (+Rap) was added where indicated. The format and data presentation were as described in Figure 1. Nucl.-Cyto., nuclear-cytoplasmic.

Figure 11

NCR-sensitive gene expression is abolished in a bmh1Δ,bmh2Δ mutant, whereas retrograde gene expression becomes constitutive. Wild-type (10560-2B) and bmh1Δ,bmh2Δ (RRY1216) cultures were grown to A_{600 nm} = 0.5 in YNB medium containing the indicated nitrogen source: glutamine (Gln), ammonia (Am.), serine (Ser.), urea, or proline (Pro). Total RNA was then isolated and Northern blots prepared as described in Materials and Methods. Blots were probed for NCR-sensitive [(A) GDH2, (B) DAL80, or (C) DAL5 probes] or retrograde [(D) CIT2] gene expression. H3 was used as the loading control. W.T., wild type.

Figure 12

Rapamycin-elicited, NCR-sensitive gene expression is abolished in bmh1Δ,bmh2Δ. Wild type (10560-2B) and bmh1Δ,bmh2Δ (RRY1216) were cultured in YNB medium containing the indicated nitrogen source. The cultures were then treated with 200 ng/ml rapamycin. Northern blots were prepared as described in Materials and Methods. Am., ammonia; Gln, glutamine; Pro, proline; Rap, rapamycin; W.T., wild type.

Figure 13

(A and B) bmh1Δ,bmh2Δ is epistatic to ure2Δ. ure2Δ (RR114), bmh1Δ,bmh2Δ (RRY1216), and ure2Δ, bmh1Δ,bmh2Δ (BS0033a) cells were cultured in YNB medium containing the indicated nitrogen sources. Total RNA was then isolated and Northern blots prepared. These blots were probed for NCR-sensitive gene expression with (A) DAL5 and (B) DAL80 probes. Am., ammonia; Gln, glutamine; Pro, proline.

Figure 14

Nuclear Gln3-GFP localization in response to rapamycin treatment requires Bmh1/2. Gln3-GFP (pRS416-GLN3-GFP) transformants of (A) wild-type (10560-2B) and (B) bmh1Δ,bmh2Δ (RRY1216) cultures were grown in YNB-glutamine medium to a cell density of A_{600 nm} = 0.5. Six samples (two images each) were collected for the zero time point and then pairs of images were collected thereafter for 60 min following rapamycin treatment. As described in the text, Gln3-GFP localization was scored as either completely cytoplasmic or nuclear-cytoplasmic. Nucl.-Cyto., nuclear-cytoplasmic.

Figure 15

Nuclear Gln3-GFP localization in response to Msx treatment is reduced in bmh1Δ,bmh2Δ. (A) Wild-type (10560-2b) and (B) bmh1Δ,bhm2Δ cells (RRY1216). The experiment was performed as described in Figure 14, except that the cells were treated with Msx for ∼25 min in place of rapamycin. Nucl.-Cyto., nuclear-cytoplasmic.

Figure 16

Gln3-Myc¹³ phosphorylation decreases in a bmh1Δ,bmh2Δ mutant. Wild-type (10560-2B, Panels A-C) and bmh1Δ,bmh2Δ (RRY1216, Panels A and C) cells were cultured to a cell density of A_{600 nm} = 0.5 in YNB-ammonia medium. Cultures were then treated with 200 ng/ml rapamycin where indicated. Extracts of all cultures were then prepared and subjected to western blot analyses as described in Materials and Methods. Rap, rapamycin; W.T., wild type.

Figure 17

Schematic summary of results obtained from experiments presented in this and earlier work. (A) Epistatic relationships of the genes encoding mTorC1, Sit4, Ure2, Gcn2, Gcn4, and Bmh1/2 in the regulation of NCR-sensitive Gln3 localization. Green arrows and designations indicate positive regulation, whereas red bars and designations indicate negative regulation. (B) Description of the global relationships of mTorC1- and Gcn2-mediated regulation on Gln3 localization and protein synthesis when cells are grown in nitrogen-replete medium.