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. 2017 Mar 31;292(13):5311-5324.
doi: 10.1074/jbc.M117.778209. Epub 2017 Feb 15.

Coordinate Regulation of Yeast Sterol Regulatory Element-binding Protein (SREBP) and Mga2 Transcription Factors

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Free PMC article

Coordinate Regulation of Yeast Sterol Regulatory Element-binding Protein (SREBP) and Mga2 Transcription Factors

Risa Burr et al. J Biol Chem. .
Free PMC article

Abstract

The Mga2 and Sre1 transcription factors regulate oxygen-responsive lipid homeostasis in the fission yeast Schizosaccharomyces pombe in a manner analogous to the mammalian sterol regulatory element-binding protein (SREBP)-1 and SREBP-2 transcription factors. Mga2 and SREBP-1 regulate triacylglycerol and glycerophospholipid synthesis, whereas Sre1 and SREBP-2 regulate sterol synthesis. In mammals, a shared activation mechanism allows for coordinate regulation of SREBP-1 and SREBP-2. In contrast, distinct pathways activate fission yeast Mga2 and Sre1. Therefore, it is unclear whether and how these two related pathways are coordinated to maintain lipid balance in fission yeast. Previously, we showed that Sre1 cleavage is defective in the absence of mga2 Here, we report that this defect is due to deficient unsaturated fatty acid synthesis, resulting in aberrant membrane transport. This defect is recapitulated by treatment with the fatty acid synthase inhibitor cerulenin and is rescued by addition of exogenous unsaturated fatty acids. Furthermore, sterol synthesis inhibition blocks Mga2 pathway activation. Together, these data demonstrate that Sre1 and Mga2 are each regulated by the lipid product of the other transcription factor pathway, providing a source of coordination for these two branches of lipid synthesis.

Keywords: SREBP; fatty acid metabolism; hypoxia; lipid metabolism; membrane transport; mga2; sre1; transcription regulation; yeast.

Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

FIGURE 1.
FIGURE 1.
mga2Δ cells have reduced Sre1N accumulation in the presence and absence of oxygen. A and C, Western blots, probed with monoclonal anti-Sre1 IgG (5B4) and polyclonal anti-Dsc5 IgG (for loading) and imaged by LI-COR Biosciences Odyssey CLx, of lysates treated with alkaline phosphatase for 1 h from WT, mga2Δ, dsc2Δ, or sre1Δ cells grown for 0 or 4 h in the absence of oxygen (A) or 6 h in the statin CPN (200 μm) or vehicle (0.12% EtOH, 400 μm NaCl) (C). P and N denote precursor and cleaved N-terminal transcription factor forms, respectively. Asterisks denote nonspecific bands. B and D, quantification from A and C of three (A and B) or four (C and D) biological replicates normalized for loading to Dsc5 and then normalized to maximum signal (WT N terminus band after treatment; lane 2) for comparison between blots. Error bars are 1 S.D. (**, p < 0.01 for N terminus by two-tailed Student's t test). Quantities of the precursor and nuclear form are stacked to give an approximation of total signal per treatment. Average percent cleavage (clvg.) is calculated by dividing the quantity of N terminus by the sum of both N terminus and precursor.
FIGURE 2.
FIGURE 2.
Defect in Sre1 cleavage in the absence of mga2 is amplified by positive feedback. A, Western blot, probed with monoclonal anti-Sre1 IgG (5B4) and polyclonal anti-Dsc5 IgG (for loading) and imaged by LI-COR Biosciences Odyssey CLx, of lysates treated with alkaline phosphatase for 1 h from WT, mga2Δ, or sre1Δ cells with WT or sre1-MP as indicated. Cells were grown for 0 or 4 h in the absence of oxygen. P and N denote precursor and cleaved N-terminal transcription factor forms, respectively. B, quantification from A of four biological replicates normalized for loading to Dsc5 and then normalized to maximum signal (WT N terminus band after treatment; lane 2) for comparison between blots. Error bars are 1 S.D. (**, p < 0.01 for N terminus by two-tailed Student's t test). Quantities of the precursor and nuclear form are stacked to give an approximation of total Sre1 signal per treatment.
FIGURE 3.
FIGURE 3.
Cleavage defect in mga2Δ cells occurs at a step downstream of Sre1-Scp1 complex formation and is not specific to Sre1. A, Western blot of cell lysates from the indicated strains grown in the presence of oxygen probed with a mixture of anti-Scp1 IgG monoclonal antibodies 8G4C11, 1G1D6, and 7B4A3 (imaged by film) or anti-Dsc5 IgG (imaged by LI-COR Biosciences Odyssey CLx). The blot is representative of four biological replicates. B, WT or mga2Δ cells expressing scp1-13xmyc and mga2Δ cells expressing untagged scp1 were grown in the presence of oxygen, and Scp1-13xMyc was immunoprecipitated (IP) from Nonidet P-40-solubilized membranes using monoclonal anti-Myc 9E10 IgG as described under “Experimental Procedures.” Input and 5-fold enriched bound fractions were analyzed by Western blotting and imaged by LI-COR Biosciences Odyssey CLx using polyclonal anti-Sre1 IgG and polyclonal anti-Myc IgG. The blot is representative of three biological replicates. C, indicated strains expressing vector (−) or scp1 (+) from a cauliflower mosaic virus promoter (17) were precultured in minimal medium lacking leucine, transferred to YES complete medium, and grown for 4 h in the presence or absence of oxygen. The Western blot was probed with anti-Sre1 IgG polyclonal antibody and imaged by LI-COR Biosciences Odyssey CLx and is representative of five biological replicates. P and N denote Sre1 precursor and N-terminal forms. Asterisks indicate nonspecific bands. D, Western blot, probed by polyclonal anti-Sre2 and polyclonal anti-Cdc48 (for loading) and imaged by LI-COR Biosciences Odyssey CLx, of lysates treated with alkaline phosphatase for 1 h from WT, mga2Δ, dsc2Δ, or sre2Δ cells grown in the presence of oxygen. E, quantification from D of six biological replicates normalized for loading to Cdc48 and then normalized to WT N terminus band for comparison between blots. Error bars are 1 S.D. (**, p < 0.01 for N terminus by two-tailed Student's t test). Quantities of the precursor and nuclear form are stacked to give an approximation of total signal per treatment. Average percent cleavage (clvg.) is calculated by dividing the quantity of N terminus by the sum of both N terminus and precursor.
FIGURE 4.
FIGURE 4.
Dsc E3 ligase is functional in mga2Δ cells. A, digitonin-solubilized membrane protein was prepared from WT, mga2Δ, or dsc2Δ cells grown in the presence or absence of oxygen for 6 h, and the Dsc E3 ligase complex was immunoprecipitated (IP) with anti-Dsc2 polyclonal IgG as described under “Experimental Procedures.” Equal quantities of input, unbound, and 10-fold bound fractions were analyzed by immunoblotting using HRP-conjugated antibodies against Dsc1, Dsc2, Dsc3, Dsc4, and Dsc5 (imaged by film). The blot is representative of three biological replicates. B, Western blot, probed with polyclonal anti-Sre1 IgG and monoclonal anti-actin (for loading) and imaged by LI-COR Biosciences Odyssey CLx, of lysates from WT, mga2Δ, dsc1Δ, or sre1Δ yeast grown for 2 h in the presence or absence of oxygen and then treated with BFA (100 μg/ml) or EtOH vehicle for another 2 h in the presence or absence of oxygen. Quantification of Sre1 precursor (Sre1-P) normalized to WT vehicle in the absence of oxygen (lane 7) is shown below. The blot is representative of two biological replicates. C, Western blot, probed with polyclonal anti-Dsc1 IgG and imaged by LI-COR Biosciences Odyssey CLx, of Nonidet P-40-solubilized membrane protein from WT, mga2Δ, or dsc2Δ cells grown in the presence of oxygen. M and I indicate mature and intermediate glycosylated forms, respectively. D, quantification of Dsc1 from C of seven replicates. The quantity of the mature form was divided by total Dsc1 signal for percent mature, allowing comparison between lanes and blots. Error bars are 1 S.D. (*, p < 0.05; **, p < 0.01 by two-tailed Student's t test).
FIGURE 5.
FIGURE 5.
mga2 deletion disrupts general membrane transport. A, anp1-GFP ost1-mCherry (WT) and mga2Δ anp1-GFP ost1-mCherry (mga2Δ) cells were cultured in the presence of oxygen and 0.9% (w/v) BSA for 6 h and imaged by confocal microscopy. These images are representative of three biological replicates. Scale bar, 5 μm. B, quantification of cells with altered Anp1-GFP localization from A of three biological replicates denoted by different marker symbols. n ≥ 150 cells per replicate. Error bars are mean ± 1 S.D. (**, p < 0.01 by χ2 analysis with Bonferroni correction). C, box-and-whisker quantification of normalized Ost1-mCherry signal intensity from A of n ≥ 300 cells from three biological replicates. Whiskers are the maximum and minimum intensity values (**, p < 0.01 by one-way ANOVA and post hoc multiple comparisons with Bonferroni correction). D, Western blot, probed by monoclonal anti-GFP IgG, polyclonal anti-mCherry IgG, and polyclonal anti-Dsc2 IgG (for loading) and imaged by LI-COR Biosciences Odyssey CLx, of lysates from anp1-GFP ost1-mCherry (WT) and mga2Δ anp1-GFP ost1-mCherry (mga2Δ) cells cultured in the presence of oxygen and 0.9% (w/v) BSA for 6 h. E, GFP quantification from D of three biological replicates represented by different marker symbols. The quantity of free GFP was divided by total GFP signal for percent GFP that is free, allowing comparison between lanes and blots. Error bars are 1 S.D. (*, p < 0.05 by paired two-tailed Student's t test, with pairing of WT and mga2Δ data on same blot due to significant interblot variance in WT signal). F, mCherry (mCh) quantification from D of four biological replicates represented by different marker symbols, normalized for loading to Anp1-GFP signal and then normalized to WT signal for comparison between blots. (n.s., no significant difference by paired two-tailed Student's t test).
FIGURE 6.
FIGURE 6.
Exogenous oleate addition rescues Sre1 cleavage and membrane trafficking in mga2Δ cells. A and B, Western blots, probed with monoclonal anti-Sre1 IgG (5B4) and polyclonal anti-Dsc5 IgG (for loading) and imaged by LI-COR Biosciences Odyssey CLx, of lysates treated with alkaline phosphatase for 1 h from WT or the indicated mutants grown for 4 h in the presence or absence of oxygen (A) or 6 h in the presence of 200 μm CPN or vehicle (0.12% EtOH, 400 μm NaCl) (B). All lanes contain 0.9% (w/v) BSA, and lanes with supplemental fatty acids conjugated to BSA are indicated. A, BSA-conjugated fatty acids were added at time 0 and were present for 4 h of growth. B, cells were grown overnight with the indicated fatty acid, washed with H2O, and then grown for 6 h with CPN or vehicle. P and N denote precursor and cleaved N-terminal transcription factor forms, respectively. Asterisks (*) indicate nonspecific bands. Blots are representative of three biological replicates. C, anp1-GFP ost1-mCherry (WT) and mga2Δ anp1-GFP ost1-mCherry (mga2Δ) cells were cultured with the indicated fatty acid supplement for 6 h and imaged by confocal microscopy. 18:X indicates supplementation of both 18:0 and 18:1. Experimental data for WT BSA and mga2Δ BSA are the same as reported in Fig. 5, A–C. Images are representative of three biological replicates. Scale bar, 5 μm. D, quantification of cells with altered Anp1-GFP localization from C of three biological replicates denoted by different marker symbols. n ≥ 150 cells per replicate. Error bars are mean ± 1 S.D. (*, p < 0.05; **, p < 0.01 by χ2 analysis with Bonferroni correction). E, box-and-whisker quantification of normalized Ost1-mCherry signal intensity from C of n ≥ 300 cells from three biological replicates. Whiskers are the maximum and minimum intensity values. (*, p < 0.05; **, p < 0.01 by one-way ANOVA and post hoc multiple comparisons with Bonferroni correction). mCh, mCherry.
FIGURE 7.
FIGURE 7.
Disrupting fatty acid homeostasis blocks general membrane transport and Sre1 cleavage. A, WT cells were grown in liquid culture in 112 nm CER or 0.1% (v/v) DMSO for 6 h. Cell density was measured by absorbance at 600 nm. Data points are the average of four biological replicates. Error bars are 1 S.D. Absorbance at time 0 among the samples ranged from 0.10 to 0.29. To focus on the difference in growth rate between conditions, data were normalized for each sample to a value of 0.2 at time point 0 before averaging. B and C, Western blots, probed with monoclonal anti-Sre1 IgG (5B4) and polyclonal anti-Dsc5 (for loading) and imaged by LI-COR Biosciences Odyssey CLx, of lysates treated with alkaline phosphatase for 1 h from WT cells. Cells were grown in 112 nm CER or 0.1% (v/v) DMSO for 2 h under normoxia. Cells were then washed, resuspended in fresh medium, and grown without cerulenin for 4 h in the presence or absence of oxygen (B and C) with the indicated BSA-conjugated fatty acid supplemented (C). P and N denote precursor and cleaved N-terminal transcription factor forms, respectively. Asterisks (*) indicate nonspecific bands. Blots are representative of two biological replicates. D, anp1-GFP ost1-mCherry cells were precultured for 1 h in the indicated BSA-conjugated fatty acid. Then 112 nm CER or 0.1% (v/v) DMSO (−CER) was added, and cells were cultured for an additional 3 h before imaging by confocal microscopy. 18:X indicates supplementation of both 18:0 and 18:1. Images are representative of three biological replicates. Scale bar, 5 μm. E, quantification of cells with altered Anp1-GFP localization from D of three biological replicates denoted by different marker symbols. n ≥ 100 cells per replicate. Error bars are mean ± 1 S.D. (**, p < 0.01 by χ2 analysis with Bonferroni correction). E, box-and-whisker quantification of normalized Ost1-mCherry signal intensity from D of n ≥ 300 cells from three biological replicates. Whiskers are the maximum and minimum intensity values (**, p < 0.01 by one-way ANOVA and post hoc multiple comparisons with Bonferroni correction). mCh, mCherry.
FIGURE 8.
FIGURE 8.
Inhibition of sterol synthesis blocks Mga2 transcription factor activity. A, Western blot, probed with monoclonal anti-FLAG M2 IgG and imaged by LI-COR Biosciences Odyssey CLx, of lysates treated with alkaline phosphatase for 1 h from WT and 2xFLAG-mga2 cells grown for 2 h in 0.1% (v/v) DMSO (D), BZ (500 μm), or CER (112 nm). P and N denote precursor and cleaved N-terminal transcription factor forms, respectively. Asterisks denote nonspecific bands. B, Western blot, probed with monoclonal anti-FLAG M2 IgG, monoclonal anti-Sre1 IgG (5B4), and polyclonal anti-Dsc5 IgG (for loading) and imaged by LI-COR Biosciences Odyssey CLx, of lysates treated with alkaline phosphatase for 1 h from WT and 2xFLAG-mga2 cells grown in the following treatments: 8 h in vehicle (0.12% EtOH, 400 μm NaCl; V), CPN (200 μm), or ITR (2 μm) or 2 h in 0.1% (v/v) DMSO (D), BZ (500 μm), or CER (112 nm). P and N denote precursor and cleaved N-terminal transcription factor forms, respectively. Asterisks denote nonspecific bands. C–E, quantitative PCR (qPCR) of the indicated genes of lysates from 2xFLAG-mga2 cells treated as indicated. Error bars are mean ± 1 S.D. of three biological replicates normalized to DMSO treatment (*, p < 0.05; **, p < 0.01 by two-tailed Student's t test).
FIGURE 9.
FIGURE 9.
Model of coordination of Mga2 and Sre1 pathways. Sterol and TAG/GPL lipid biosynthetic pathways are positively regulated by the transcription factors Sre1 and Mga2, respectively. Both Sre1 and Mga2 are product-inhibited, Sre1 by sterols and Mga2 by UFA. Mga2 regulates Sre1 through the requirement for UFA, and Sre1 regulates Mga2 through the requirement of sterols for Mga2 activity.

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