Simplified diagrammatic representation, based on data in the literature, of the two possible pathways responsible for nitrogen-responsive GATA factor regulation. This poses the question, Where does Msx function as an inhibitor, at the top of the TorC1 pathway (Pathway 2) or in a separate pathway (Pathway 1)? Green colors in arrows or molecule designations are used to indicate that a molecule is active or a reaction is facilitated. Red colors, on the other hand, indicate inactive molecules or inhibition of a reaction. Phosphorylation of GATA factors and Ure2 are those associated with Pathway 2. No specific molecular models related to Msx-elicited phosphorylation have been proposed.

Transcription of NCR-sensitive Genes GDH2, GAT1, DAL80, and DAL5 in response to Msx and rapamycin. Total RNA was isolated from wild type (TB50), gln3Δ (FV005), and gat1Δ (FV006) cells grown in YNB-ammonia medium and treated with rapamycin (+Rap) or methionine sulfoximine (+Msx). Control cells were similarly grown but untreated (Am.). mRNA levels were quantified by quantitative RT-PCR as described under “Materials and Methods.” GDH2 (panel A), GAT1 (panel B), DAL80 (panel C), and DAL5 (panel D) values were normalized with TBP1.

Localization of Gln3-Myc¹³ and Gat1-Myc¹³ in response to Msx and rapamycin. Wild type (TB123), pph21Δpph22Δ (03705d), or sit4Δ (TB136-2a) strains, containing Gln3-Myc¹³ (panels A and B) or wild type (FV063), pph21Δpph22Δ (03879c), or sit4Δ (FV066) strains containing Gat1-Myc¹³ (panels C and D) were grown in YNB-ammonia medium. The cultures were sampled for indirect immunofluorescence assay of Gln3-Myc¹³ or Gat1-Myc¹³ localization (Am.). Rapamycin (+Rap) or methionine sulfoximine (+Msx) was then added, and the cultures were sampled again 20 and 30 min later, respectively. Indirect immunofluorescence assays were performed and imaged as described under “Materials and Methods.” For each histogram, cells were scored for intracellular Gln3-Myc¹³ or Gat1-Myc¹³ localization (cytoplasmic, red bars, nuclear-cytoplasmic, yellow bars; nuclear, green bars) using criteria described under “Materials and Methods.” Multiple examples of images in each category may be found in of Tate et al. (). Images from which the histograms were derived are shown above each histogram. The upper member of each pair (panels A and C) depicts red Gln3-Myc¹³- or Gat1-Myc¹³-derived fluorescence, and the lower one shows DAPI-positive material fluorescence. The precision of the IF assays we employ has been analyzed in detail and is described under “Materials and Methods.”

Gln3-Myc¹³ and Gat1-Myc¹³ binding to the GDH2 and DAL5 promoters in response to Msx and rapamycin. Cells were grown in YNB-ammonia medium and treated with rapamycin (+Rap) or methionine sulfoximine (+Msx). Control cells were similarly grown but untreated (Am.). 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 specific for the GDH2 promoter (GDH2P) or for the DAL5 promoter (DAL5P) and for a region 2.5 kb upstream of the DAL5 open reading frame as a control (DAL5U). For each immunoprecipitation, IP/IN values were calculated as ([GDH2P]^IP/[GDH2P]^IN − [DAL5U]^IP/[DAL5U]^IN). Panel A, Gln3-Myc¹³ binding to P_GDH2 is shown. Untagged wild type (TB50), GLN3-MYC¹³ wild type (TB123), and GLN3-MYC¹³ gat1Δ (FV018) cells were subjected to ChIP analysis. Panel B, Gat1-Myc¹³ binding to P_GDH2 is shown. Untagged wild type (TB50), GAT1-MYC¹³ wild type (FV063), and GAT1-MYC¹³ gln3Δ (FV064) cells were subjected to ChIP analysis. Panel C, Gln3-Myc¹³ binding to P_DAL5 is shown. Untagged wild type (TB50), GLN3-MYC¹³ wild type (TB123), and GLN3-MYC¹³ gat1Δ (FV018) cells were subjected to ChIP analysis. Panel D, Gat1-Myc¹³ binding to P_DAL5 is shown. Untagged wild type (TB50), GAT1-MYC¹³ wild type (FV063), and GAT1-MYC¹³ gln3Δ (FV064) cells were subjected to ChIP analysis.

Phosphatase requirements for transcription of NCR-sensitive genes GDH2 and DAL5 in response to Msx and rapamycin. Total RNA was isolated from wild type (TB50), sit4Δ (FV029), and pph21Δpph22Δ (FV239) cells grown in YNB-ammonia medium and treated with rapamycin (+Rap) or methionine sulfoximine (+Msx). Control cells were similarly grown but untreated (Am.). GDH2 (panel A) and DAL5 (panel B) mRNA levels were quantified by quantitative RT-PCR as described in .

Localization of Gln3-GFP and Gat1-GFP in wild type and PP2A (pph21Δpph22Δ) mutant strains in response to Msx. Wild type (FV063; panels A, B, E, and F) or pph21Δpph22Δ (03879c; panels C, D, G, and H) mutant strains containing a chromosomal copy of Gat1-Myc¹³ were transformed with CEN-plasmids pRS416-Gln3-GFP or pRS416-Gat1-GFP, and transformants were grown in YNB-ammonia medium. Six samples from each culture were successively collected for real time imaging as described under “Materials and Methods.” Msx was then added, and sample collection was continued thereafter as indicated. Microscopic images in panels A, C, E, and G were derived from images used to determine intracellular Gat1-GFP localization in the histograms of panels B, D, F, and H. Times that appear on the abscissa are the averages of the two times at which the pairs of images generated from each sample were completed after the addition of methionine sulfoximine to the cultures. For each histogram, cells were scored for intracellular Gat1-GFP localization using the three category method (cytoplasmic, red bars; nuclear-cytoplasmic, yellow bars; nuclear, green bars) as described under “Materials and Methods.” Due to weak signals and the high background fluorescence, it is not possible to confidently distinguish cells in which Gln3-GFP is nuclear from those where it is nuclear-cytoplasmic (). Therefore, only two category scoring was performed; Gln3-GFP was scored as completely cytoplasmic or nuclear-cytoplasmic. Error bars in histograms derived from untreated cells designate 1 S.D. relative to the average of the data collected. Error bars do not appear in the case of data obtained from treated cells because the response time courses modestly vary (plus or minus a few minutes) from day to day. Normalization of the shapes of the curves, which we are unable to do, would be required to present statistical analysis of the data. It is the qualitative responses of the treatments and their overall time courses that we feel is most important.

Localization of Gln3-GFP and Gat1-GFP in wild type and sit4Δ mutant strains in response to Msx. The experimental format and data presentation were the same as those in except for the strains used as transformation recipients. In this experiment they were wild type (TB50; panels A, B, E, and F) and sit4Δ (FV029; panels C, D, G, and H) containing an untagged chromosomal copy of GAT1.

Localization of Gln3-GFP and Gat1-GFP in wild type and pph21Δpph22Δ mutant strains in response to rapamycin. The experimental format and data presentation were the same as those in with two exceptions. First, the strains used as transformation recipients were wild type (TB50; panels A, B, E, and F) and pph21Δpph22Δ (FV239; panels C, D, G, and H). Second, rapamycin replaced methionine sulfoximine as the inhibitor added to the cultures.

Localization of Gln3-GFP and Gat1-GFP in wild type and sit4Δ mutant strains in response to rapamycin. The experimental format and data presentation were the same as those in with two exceptions. First, the strains used as transformation recipients were wild type (TB50; panels A, B, E, and F) and sit4Δ (FV029; panels C, D, G, and H). Second, rapamycin replaced methionine sulfoximine as the inhibitor added to the cultures.

Phosphatase requirements for Gln3-Myc¹³ and Gat1-Myc¹³ binding to the GDH2 and DAL5 promoters promoter in response to Msx and rapamycin. Cells were grown in YNB-ammonia medium and treated with rapamycin (+Rap) or methionine sulfoximine (+Msx). Control cells were similarly grown but untreated (Am.). ChIP was performed as described in . Panel A, Gln3-Myc¹³ binding to P_GDH2 is shown. GLN3-MYC¹³ wild type (TB123), GLN3-MYC¹³ sit4Δ (TB136-2a), and GLN3-MYC¹³ pph21Δpph22Δ (FV221) cells were subjected to ChIP analysis. Panel B, Gat1-Myc¹³ binding to P_GDH2 is shown. GAT1-MYC¹³ wild type (FV063), GAT1-MYC¹³ sit4Δ (FV066), and GAT1-MYC¹³ pph21Δpph22Δ (03879c) cells were subjected to ChIP analysis. Panel C, Gln3-Myc¹³ binding to P_DAL5 is shown. GLN3-MYC¹³ wild type (TB123), GLN3-MYC¹³ sit4Δ (TB136-2a), and GLN3-MYC¹³ pph21Δpph22Δ (FV221) cells were subjected to ChIP analysis. Panel D, Gat1-Myc¹³ binding to P_DAL5 is shown. GAT1-MYC¹³ wild type (FV063), GAT1-MYC¹³ sit4Δ (FV066), and GAT1-MYC¹³ pph21Δpph22Δ (03879c) cells were subjected to ChIP analysis.