Tor pathway control of the nitrogen-responsive DAL5 gene bifurcates at the level of Gln3 and Gat1 regulation in Saccharomyces cerevisiae

Abstract

The Tor1,2 protein kinases globally influence many cellular processes including nitrogen-responsive gene expression that correlates with intracellular localization of GATA transcription activators Gln3 and Gat1/Nil1. Gln3-Myc(13) and Gat1-Myc(13) are restricted to the cytoplasm of cells provided with good nitrogen sources, e.g. glutamine. Following the addition of the Tor1,2 inhibitor, rapamycin, both transcription factors relocate to the nucleus. Gln3-Myc(13) localization is highly dependent upon Ure2 and type 2A-related phosphatase, Sit4. Ure2 is required for Gln3 to be restricted to the cytoplasm of cells provided with good nitrogen sources, and Sit4 is required for its location to the nucleus following rapamycin treatment. The paucity of analogous information concerning Gat1 regulation prompted us to investigate the effects of deleting SIT4 and URE2 on Gat1-Myc(13) localization, DNA binding, and NCR-sensitive transcription. Our data demonstrate that Tor pathway control of NCR-responsive transcription bifurcates at the regulation of Gln3 and Gat1. Gat1-Myc(13) localization is not strongly influenced by deleting URE2, nor is its nuclear targeting following rapamycin treatment strongly dependent on Sit4. ChIP experiments demonstrated that Gat1-Myc(13) can bind to the DAL5 promoter in the absence of Gln3. Gln3-Myc(13), on the other hand, cannot bind to DAL5 in the absence of Gat1. We conclude that: (i) Tor pathway regulation of Gat1 differs markedly from that of Gln3, (ii) nuclear targeting of Gln3-Myc(13) is alone insufficient for its recruitment to the DAL5 promoter, and (iii) the Tor pathway continues to play an important regulatory role in NCR-sensitive transcription even after Gln3-Myc(13) is localized to the nucleus.

FIGURE 1.

Effect of deleting type 2A-related phosphatase genes SIT4 and PPH3 on rapamycin-induced DAL5 expression. Total RNA was isolated from wild type (TB123), pph3Δ (FV003), sit4Δ (TB136-2a), and pph3Δ sit4Δ (FV004) cells expressing GLN3-MYC¹³ that replaced the native GLN3 gene. Cells were grown in YNB-glutamine medium and treated with rapamycin (Rap) (0.2 μg/ml) for 30 min. Control cells were similarly grown but untreated. DAL5 mRNA levels were quantified by quantitative RT-PCR, as described under “Materials and Methods.” DAL5 values were normalized with TBP1. The values represent the averages of at least three experiments from independent cultures, and the error bars indicate standard errors. 30 μg of total RNA from each sample were subjected to Northern blot analysis. HHT1 was used as the loading and transfer efficiency control.

FIGURE 2.

A, relative contributions of Gat1 and Gln3 to rapamycin-induced DAL5 expression. Total RNA was isolated from wild type (TB50), gat1Δ (FV006), gln3Δ (FV005), sit4Δ (FV029), gat1Δsit4Δ (FV008), and gln3Δsit4Δ (FV030) cultures grown in YNB-glutamine medium. The cells were treated and analyses performed as in Fig. 1. B, functionality and normal regulation of the integrated GAT1-MYC¹³ construct. Total RNA was isolated from wild type (TB50) and wild type GAT1-MYC¹³ (FV063) cells grown in glutamine (Gln) medium in the presence or absence of 0.2 μg/ml rapamycin (Rap) for 30 min, 60 min after transfer from glutamine to proline (shift Pro), or nitrogen-free medium (shift -N), proline (Pro), or ammonium (Am) medium in the presence or absence of 2 mm methionine sulfoximine (Msx) for 20 min. The cells were treated and analyses performed as in Fig. 1. W.T., wild type.

FIGURE 3.

Effects of rapamycin on the intracellular localization of Gat1-Myc¹³ in wild type, sit4Δ, and pph3Δ strains. Wild type (W.T.) and mutant strains were grown in YNB-glutamine medium. Split cultures were left untreated (Gln) or treated with rapamycin (0.2 μg/ml) for 20 min (+Rap), sampled, and processed for immunofluorescence microscopy as described under “Materials and Methods.” Strain numbers appear below the pertinent genotype. The images are presented in pairs with Gat1-Myc¹³-dependent fluorescence above and DAPI-stained cells below. The images and corresponding histograms below them were taken from the same slides. Intracellular distributions of Gat1-Myc¹³ (using criteria described under “Materials and Methods”) are indicated by the bar color in the histograms: red, cytoplasmic; yellow, nuclear-cytoplasmic; green, nuclear.

FIGURE 4.

A and B, effects of sit4Δ, gln3Δ, and gat1Δ on rapamycin-induced binding of Gat1-Myc¹³ and Gln3-Myc¹³ to the DAL5 promoter. C and D, effect of deleting GAT1 on Gln3-Myc¹³ localization. Wild type (W.T.) untagged (TB50), wild type GAT1-MYC¹³ (FV063), gln3Δ GAT1-MYC¹³ (FV064), and sit4Δ GAT1-MYC¹³ (FV066) (A), and wild type untagged (TB50), wild type GLN3-MYC¹³ (TB123), gat1Δ GLN3-MYC¹³ (FV018), sit4Δ GLN3-MYC¹³ (TB136-2a) (B) strains were grown in YNB-glutamine medium with or without the addition of rapamycin (0.2 μg/ml) for 30 min. ChIP was performed using antibodies against c-Myc as described under “Materials and Methods.” Quantitative PCR of IP and IN fractions was performed with primers for DAL5 promoter (DAL5P) and for a region 2.5 kb upstream of DAL5 open reading frame as a control (DAL5U). For each immunoprecipitation, IP/IN values were calculated as follows: [DAL5P]^IP/[DAL5P]^IN – [DAL5U]^IP/[DAL5U]^IN, normalized to the value obtained with wild type-induced cells. Histograms represent the average of at least two experiments from independent cultures. The error bars indicate standard errors. C and D, wild type and gat1Δ strains were grown in YNB-glutamine medium. Split cultures were left untreated (Gln) or treated with rapamycin (0.2 μg/ml) for 20 min (+Rap), sampled, and processed for immunofluorescence microscopy as described under “Materials and Methods” and in the legend to Fig. 3.

FIGURE 5.

Effects of sit4Δ, ure2Δ, and sit4Δure2Δ mutations on DAL5 expression. Total RNA was isolated from TB wild type (TB123), sit4Δ (TB136-2a) ure2Δ (TB138-1a), and ure2Δsit4Δ (FV072) cells grown in YNB-glutamine medium that were untreated or treated with 0.2 μg/ml rapamycin for 30 min. The cells were treated, and analyses were performed as for Fig. 1. W.T., wild type.

FIGURE 6.

Effects of rapamycin treatment on the intracellular localization of Gln3-Myc¹³ and Gat1-Myc¹³ in ure2Δ, sit4Δ, and ure2Δsit4Δ mutant strains. The formats for the experiments and presentation of the data were the same as in Fig. 3. A and B, Gln3-Myc¹³ was visualized. C and D, Gat1-Myc¹³ was visualized. Note that FV071 and FV072 have the same genotypes and were constructed in the same genetic background (see Table 1). In a similar experiment, FV072 gave results similar to those depicted here for FV071. W.T., wild type.

FIGURE 7.

ChIP analysis of rapamycin-induced recruitment of Gln3-Myc¹³ and Gat1-Myc¹³ to the DAL5 promoters in wild type, sit4Δ, ure2Δ, and ure2Δsit4Δ strains. Wild type untagged (TB50), wild type GLN3-MYC¹³ (TB123), ure2Δ GLN3-MYC¹³ (TB138-1a), sit4Δ GLN3-MYC¹³ (TB136-2a), ure2Δsit4Δ GLN3-MYC¹³ (FV072), wild type GAT1-MYC¹³ (FV063), ure2Δ GAT1-MYC¹³ (FV088), sit4Δ GAT1-MYC¹³ (FV066), and ure2Δsit4Δ GAT1-MYC¹³ (FV089) strains were grown in YNB-glutamine medium with or without addition of rapamycin (0.2 μg/ml) for 30 min. ChIP and subsequent quantitative PCR were performed as in Fig. 4. W.T., wild type.

FIGURE 8.

Diagrammatic summary of data, showing bifurcation of Tor pathway at the level of GATA factor regulation. The arrows and bars indicate positive and negative regulation, respectively. The absence of arrows or bars indicates insufficient data are available to make such a characterization. This diagrammatic summary does not address the molecular mechanism of Gln3 regulation by Ure2 (two models have been suggested (8, 11)) or the transfer of environmental signals to Tor1,2 and other protein kinases.