Distinct phosphatase requirements and GATA factor responses to nitrogen catabolite repression and rapamycin treatment in Saccharomyces cerevisiae

Abstract

In yeast, rapamycin (Rap)-inhibited TorC1, and the phosphatases it regulates (Sit4 and PP2A) are components of a conserved pathway regulating the response of eukaryotic cells to nutrient availability. TorC1 and intracellular nitrogen levels regulate the localization of Gln3 and Gat1, the activators of nitrogen catabolite repression (NCR)-sensitive genes whose products are required to utilize poor nitrogen sources. In nitrogen excess, Gln3 and Gat1 are cytoplasmic, and NCR-sensitive transcription is repressed. During nitrogen limitation or Rap treatment, Gln3 and Gat1 are nuclear, and transcription is derepressed. We previously demonstrated that the Sit4 and Pph21/22-Tpd3-Cdc55/Rts1 requirements for nuclear Gln3 localization differ. We now show that Sit4 and Pph21/22-Tpd3-Cdc55/Rts1 requirements for NCR-sensitive and Rap-induced nuclear Gat1 localization markedly differ from those of Gln3. Our data suggest that Gln3 and Gat1 localizations are controlled by two different regulatory pathways. Gln3 localization predominantly responds to intracellular nitrogen levels, as reflected by its stronger NCR-sensitivity, weaker response to Rap treatment, and strong response to methionine sulfoximine (Msx, a glutamine synthetase inhibitor). In contrast, Gat1 localization predominantly responds to TorC1 regulation as reflected by its weaker NCR sensitivity, stronger response to Rap, and immunity to the effects of Msx. Nuclear Gln3 localization in proline-grown (nitrogen limited) cells exhibits no requirement for Pph21/22-Tpd3/Cdc55, whereas nuclear Gat1 localization under these conditions is absolutely dependent on Pph21/22-Tpd3/Cdc55. Furthermore, the extent to which Pph21/22-Tpd3-Cdc55 is required for the TorC1 pathway (Rap) to induce nuclear Gat1 localization is regulated in parallel with Pph21/22-Tpd3-Cdc55-dependent Gln3 dephosphorylation and NCR-sensitive transcription, being highest in limiting nitrogen and lowest when nitrogen is in excess.

FIGURE 1.

Time course, scoring, and precision of methods used to assay rapamycin-induced nuclear Gat1-GFP localization. Panel A, five samples (S-1 to S-5) were taken before rapamycin addition with two images collected from each sample as described under “Materials and Methods.” Thereafter, rapamycin was added to the glutamine-grown wild type (FV063) culture, and sampling (10 samples, S-6 to S-15) was continued. Electronic acquisition of two images per sample was, respectively, completed at the times indicated on the abscissa. Spacing between results from the pairs of images in the histogram was increased to indicate this. For example, the 3.0- and 4.0-min time points were derived from the same culture sample. The histograms are color-coded as described under “Materials and Methods.” Panel B, examples of cells scored by the criteria outlined under “Materials and Methods.” Images A and B, cytoplasmic; images C and D, nuclear cytoplasmic (NC); images E and F, nuclear (N). Panel C, co-localization of Gat1-GFP and DAPI-positive signals in three pairs of images (H and I, J and K, and L and M) is shown. Panel D, day-to-day, transformant-to-transformant precision of the scoring method is shown. Each bar of the histogram derives from cells scored in the images (2 each) of three samples derived from three independent transformants; error bars indicate the S.D. of these data, n = 9 samples. The absence of error bars indicates the S.D. was 0.

FIGURE 2.

Requirements of the catalytic subunits of Sit4 and Pph21/22-Tpd3-Cdc55/Rts1 protein phosphatases for intracellular Gat1-GFP localization in untreated and rapamycin-treated (+Rap), glutamine-grown wild type (FV063), sit4Δ (FV066), pph21Δpph22Δ (03879c), sit4Δpph21Δ (03827c), and sit4Δpph22Δ (03839c) mutant cells. Microscopic images in panels A and C derived from images used to determine intracellular Gat1-GFP localization in the histograms of panels B and D. Times that appear on the abscissa are the averages of the two times at which the pairs of images from each sample were completed after the addition of rapamycin to the culture.

FIGURE 3.

Requirements of the regulatory subunits of Pph21/22-Tpd3-Cdc55/Rts1 for intracellular Gat1-GFP localization in untreated and rapamycin-treated, glutamine-grown wild type (FV063), pph21Δpph22Δ (03879c), tpd3Δ (FV210), cdc55Δ (FV207), rts1Δ (FV208), and cdc55Δrts1Δ (FV226) mutant cells. The experimental format and presentation of data are the same as in Fig. 2.

FIGURE 4.

Requirements of the catalytic subunits of Sit4 and Pph21/22-Tpd3-Cdc55/Rts1 protein phosphatases for intracellular Gat1-GFP localization in untreated and rapamycin-treated, proline-grown wild type (FV063), sit4Δ (FV066), pph21Δpph22Δ (03879c), sit4Δpph21Δ (03827c), and sit4Δpph22Δ (03839c) mutant cells. The experimental format and presentation of data are the same as in Fig. 2.

FIGURE 5.

Requirements of the Pph21/22-Tpd3-Cdc55/Rts1 regulatory subunits for intracellular Gat1-GFP localization in untreated and rapamycin-treated, proline-grown wild type (FV063), pph21Δpph22Δ (03879c), tpd3Δ (FV210), cdc55Δ (FV207), rts1Δ (FV208), and cdc55Δrts1Δ (FV226) mutant cells. The experimental format and presentation of data are the same as in Fig. 2.

FIGURE 6.

Influence of the nitrogen source on intracellular Gat1-GFP localization in untreated and rapamycin-treated wild type (FV063) and pph21Δpph22Δ (03879c) mutant cells. The nitrogen source provided is indicated above each pair of panels. Concentration of these nitrogen sources was 0.1% unless indicated otherwise.

FIGURE 6.

Influence of the nitrogen source on intracellular Gat1-GFP localization in untreated and rapamycin-treated wild type (FV063) and pph21Δpph22Δ (03879c) mutant cells. The nitrogen source provided is indicated above each pair of panels. Concentration of these nitrogen sources was 0.1% unless indicated otherwise.

FIGURE 7.

Gln3-GFP and Gat1-GFP localizations respond oppositely under multiple conditions. Two aliquots of a wild type (FV063), sit4Δ (FV066), or pph21Δpph22Δ (03879c) culture were transformed with pRS416-Gat1-GFP and pRS416-Gln3-GFP (42, 52). Transformants were grown under identical conditions and inoculated as soon as possible into YNB-serine (Ser), glutamine (−Gln) −, or − proline (−Pro) medium. At an A_{600 nm} of 0.45–0.5, the respective cultures were sampled 6 times, and then where indicated, rapamycin (200 ng/ml) or methionine sulfoximine (2 mm Sigma) was added. The treated cultures were sampled for an additional 30–35 min. All images were derived from samples in which GATA factor localization had become stable as described under “Materials and Methods.” The black and white set points (Photoshop Levels Image Adjustment) for Gln3-GFP images required greater alteration than those of Gat1-GFP due to the high ratio of background to intracellular fluorescence that occurs due to the low concentration of Gln3 when the gene is expressed from its own promoter. Gamma settings were not altered. Two images are presented for each condition. Columns A and B depict Gln3-GFP and C and D depict Gat1-GFP.

FIGURE 8.

Electrophoretic mobility of Gat1-Myc¹³ in wild type (FV063), pph21Δpph22Δ (03879c), and sit4Δ (FV066) in untreated and rapamycin-treated (+Rap) cells. The nitrogen source provided in each of the cultures is indicated to the right of the images. The bottom images in panels A and B are overexposures of the upper images. They are presented to permit determination of whether minor low mobility bands existed in lanes 1, 4, and 5 relative to those in lanes 2 and 3. Black dots between lanes 2 and 3 of panel A highlight species that increase in the pph21Δpph22Δ mutant extract relative to wild type. The gel depicted in C was electrophoresed for a longer time than the one in A. The left image of C is a digital photograph similar to those in A, B, D, and E. The right image of C is a scan of the x-ray film photographed in the left panel.

FIGURE 9.

Panel A, summary of the Sit4 and Pph21/22-Tpd3-Cdc55/Rts1 requirements for the intracellular localization of Gln3 and Gat1 in response to the cell nitrogen supply and rapamycin treatment. Black solid and gray dashed lines indicate strong and weak regulation, respectively. Similarly, black and gray letters indicate strong and weak phosphatase requirements, respectively. Arrows and bars indicate positive and negative regulation, respectively. Ure2 regulation of the GATA factors has been included in the figure to emphasize that its negative regulation of Gln3 is stronger than that of Gat1 (48), which correlates with the NCR-sensitivity of the two GATA factor localizations. Panel B, depiction of the relative requirements of Pph21/22 for Gln3 and Gat1 localization as a function of the nitrogen source provided. The overall inverse relationship of these requirements is clearly visible. It is important to emphasize that this is a rough qualitative rather than quantitative representation. Data for the construction of this panel were taken from the present work (Gat1) and that appearing earlier for Gln3 (40, 41, 43, 46, 47, 48).