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. 2014 Jun;3(3):271-87.
doi: 10.1002/mbo3.168. Epub 2014 Mar 18.

Components of Golgi-to-vacuole trafficking are required for nitrogen- and TORC1-responsive regulation of the yeast GATA factors

Mohammad Fayyadkazan  1 , Jennifer J Tate, Fabienne Vierendeels, Terrance G Cooper, Evelyne Dubois, Isabelle Georis
Affiliations

Components of Golgi-to-vacuole trafficking are required for nitrogen- and TORC1-responsive regulation of the yeast GATA factors

Mohammad Fayyadkazan et al. Microbiologyopen. 2014 Jun.

Abstract

Nitrogen catabolite repression (NCR) is the regulatory pathway through which Saccharomyces cerevisiae responds to the available nitrogen status and selectively utilizes rich nitrogen sources in preference to poor ones. Expression of NCR-sensitive genes is mediated by two transcription activators, Gln3 and Gat1, in response to provision of a poorly used nitrogen source or following treatment with the TORC1 inhibitor, rapamycin. During nitrogen excess, the transcription activators are sequestered in the cytoplasm in a Ure2-dependent fashion. Here, we show that Vps components are required for Gln3 localization and function in response to rapamycin treatment when cells are grown in defined yeast nitrogen base but not in complex yeast peptone dextrose medium. On the other hand, Gat1 function was altered in vps mutants in all conditions tested. A significant fraction of Gat1, like Gln3, is associated with light intracellular membranes. Further, our results are consistent with the possibility that Ure2 might function downstream of the Vps components during the control of GATA factor-mediated gene expression. These observations demonstrate distinct media-dependent requirements of vesicular trafficking components for wild-type responses of GATA factor localization and function. As a result, the current model describing participation of Vps system components in events associated with translocation of Gln3 into the nucleus following rapamycin treatment or growth in nitrogen-poor medium requires modification.

Keywords: GATA factor; nitrogen availability; rapamycin; vesicular trafficking; yeast.

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Figures

Figure 1
Figure 1
Class C and D Vps proteins requirements for efficient transcription of the Gat1-activated DAL5 gene following rapamycin treatment or transferring cells from ammonia to proline medium in YNB-grown cells. Total RNA was isolated from WT (TB50), gln3Δ (FV005), gat1Δ (FV006), the Class C pep3Δ (MK46), pep5Δ (MK24), and vps16Δ (FV735) and the Class D vps3Δ (MK23), vps34Δ (FV391), and vps45Δ (FV643) mutant cells grown in YNB-ammonia medium (Am.) and treated with rapamycin (+Rap) or transferred to proline medium (shift Pro). DAL5 mRNA levels were quantified by quantitative RT-PCR as described in “Experimental Procedures.” The values reported represent the averages of at least two experiments from independent cultures; error bars indicate standard errors.
Figure 2
Figure 2
Gln3-Myc13 and Gat1-Myc13 binding to the DAL5 promoter in WT, gln3Δ and gat1Δ cells as well as in the Class C pep5Δ mutant and the Class D vps3Δ, vps34Δ, and vps45Δ mutant cells in response to rapamycin treatment or transferring cells from YNB-ammonia to YNB-proline medium. Untagged WT (TB50), GLN3-MYC13 WT (FV250), GLN3-MYC13 gat1Δ (FV018), GLN3-MYC13 pep5Δ (MK30), GLN3-MYC13 vps3Δ (MK27), GLN3-MYC13 vps34Δ (FV390), GLN3-MYC13 vps45Δ (FV644), GAT1-MYC13 WT (FV063), GAT1-MYC13 gln3Δ (FV064), GAT1-MYC13 pep5Δ (FV640), GAT1-MYC13 vps3Δ (FV641), GAT1-MYC13 vps34Δ (FV392), and GAT1-MYC13 vps45Δ (FV642) cells were grown in YNB-ammonia medium (Am.) and treated with rapamycin (+Rap) or transferred to proline medium (shift Pro). ChIP was performed using antibodies against c-myc as described in “Experimental Procedures.” qPCR of IP and IN fractions was performed with primers specific 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 follows: ([DAL5P]IP/[DAL5P]IN − [DAL5U]IP/[DAL5U]IN). The values reported represent the averages of two immunoprecipitations performed in at least two experiments from independent cultures; error bars indicate standard errors.
Figure 3
Figure 3
Requirements of Class C and D Vps proteins for intracellular Gat1-Myc13 localization in response to rapamycin or transferring cells from YNB-ammonia to YNB-proline medium. GAT1-MYC13 WT (FV063), Class C pep3Δ (FV732), pep5Δ (FV640), and vps16Δ (FV734) and Class D vps3Δ (FV641), vps34Δ (FV392) ,and vps45Δ (FV642) mutant cells were grown in YNB-ammonia medium (Am.). Cells were treated with rapamycin (+Rap) or transferred to proline medium (shift Pro). The cultures were sampled for indirect immunofluorescence assay of Gat1-Myc13 localization. Indirect immunofluorescence assays were performed and imaged as described in Experimental Procedures. Images from which the histograms were derived are displayed on the left hand side of the figure. The upper member of each pair depicts green Gat1-Myc13-derived fluorescence and the lower one shows DAPI-positive material fluorescence. For each histogram, displayed at the right hand side of the figure, cells were scored for intracellular Gat1-Myc13 localization (cytoplasmic red bars, nuclear–cytoplasmic, yellow bars; nuclear, green bars) using criteria described in Experimental Procedures. When no histogram bar is visible on the graph, that is because there were no cells found in scoring category considered. The values reported represent the averages of at least two experiments from independent cultures.
Figure 4
Figure 4
Requirements of Class C and D Vps proteins for intracellular Gln3-Myc13 localization in response to rapamycin or transferring cells from YNB-ammonia to YNB-proline medium. GLN3-MYC13 WT (FV250), Class C pep3Δ (FV733), pep5Δ (MK30), and vps16Δ (FV736) and Class D vps3Δ (MK27), vps34Δ (FV390), and vps45Δ (FV644) mutant cells were grown in YNB-ammonia medium (Am.) and treated with rapamycin (+Rap) or transferred to proline medium (shift Pro). The experimental format and data presentation are the same as those in Figure 3.
Figure 5
Figure 5
Requirements of Class C and D Vps proteins for intracellular Gln3-Myc13 and Gat1-Myc13 localization in YPD growth conditions. GLN3-MYC13 WT (FV250), pep5Δ (MK30), vps34Δ (FV390), GAT1-MYC13 WT (FV063), pep5Δ (FV640), and vps34Δ (FV392) cells were grown in YPD medium and treated with rapamycin (+Rap) or transferred to proline medium (shift Pro). The experimental format and data presentation are the same as those in Figure 3 with one exception that all the strains were grown in YPD medium instead of YNB-ammonia.
Figure 6
Figure 6
Gat1-Myc13 binding to the DAL5 promoter in WT and vps34Δ strains in response to rapamycin or transfer of YPD-grown cells to YNB-proline medium. Untagged WT (TB50), GAT1-MYC13 WT (FV063) and GAT1-MYC13 vps34Δ (FV392) cells were grown in YPD medium and treated with rapamycin (+Rap) or transferred to proline medium (shift Pro). ChIP was performed as described in Figure 2.
Figure 7
Figure 7
Class C and D Vps protein requirements for transcription of the GATA factor-activated genes DAL5, GAP1 and MEP2 in response to rapamycin or transfer of YPD-grown cells to YNB-proline medium. The strains, experimental format and data presentation are the same as those in Figure 1 with one exception that all the strains were grown in YPD medium instead of YNB-ammonia. (A) DAL5. (B) GAP1. (C) MEP2.
Figure 8
Figure 8
Subcellular fractionation of Gat1-Myc13 and Gln3-Myc13. Cell-free lysates from YPD-grown GAT1-MYC13 WT (FV063; A and C) and GLN3-MYC13 WT (FV250; B and D) cells were subjected to differential centrifugation to yield low-speed pellet (P13), supernatant (S13), high-speed pellet (P100), and soluble (S100) fractions. Equal cell equivalents were examined by Western blot to detect Gln3-Myc13, Gat1-Myc13, Pep12, and Pgk1. Protein extracts were prepared using a lysis buffer lacking NaCl (A and B) or containing 0.15 mol/L NaCl (C and D).
Figure 9
Figure 9
Epistatic Relation between Ure2 and Vps3. WT (TB50), ure2Δ (OK01), vps3Δ (MK23), ure2Δvps3Δ (08047c) cells were grown in YPD medium and treated with rapamycin (+Rap) or transferred to proline medium (shift Pro). DAL5 mRNA levels were quantified by quantitative RT-PCR as described in “Experimental Procedures.” The experimental format and data presentation are the same as those in Figure 1.
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