4-Methyl sterols regulate fission yeast SREBP-Scap under low oxygen and cell stress

Abstract

In fission yeast, orthologs of mammalian SREBP and Scap, called Sre1 and Scp1, monitor oxygen-dependent sterol synthesis as a measure of cellular oxygen supply. Under low oxygen conditions, sterol synthesis is inhibited, and Sre1 cleavage is activated. However, the sterol signal for Sre1 activation is unknown. In this study, we characterized the sterol signal for Sre1 activation using a combination of Sre1 cleavage assays and gas chromatography sterol analysis. We find that Sre1 activation is regulated by levels of the 4-methyl sterols 24-methylene lanosterol and 4,4-dimethylfecosterol under conditions of low oxygen and cell stress. Both increases and decreases in the level of these ergosterol pathway intermediates induce Sre1 proteolysis in a Scp1-dependent manner. The SREBP ortholog in the pathogenic fungus Cryptococcus neoformans is also activated by high levels of 4-methyl sterols, suggesting that this signal for SREBP activation is conserved among unicellular eukaryotes. Finally, we provide evidence that the sterol-sensing domain of Scp1 is important for regulating Sre1 proteolysis. The conserved mutations Y247C, L264F, and D392N in Scp1 that render Scap insensitive to sterols cause constitutive Sre1 activation. These findings indicate that unlike Scap, fission yeast Scp1 responds to 4-methyl sterols and thus shares properties with mammalian HMG-CoA reductase, a sterol-sensing domain protein whose degradation is regulated by the 4-methyl sterol lanosterol.

Figure 1. The S. pombe ergosterol biosynthesis pathway

Oxygen requiring enzymes are boxed. The structures of 4-methyl sterol intermediates (24-methylene lanosterol, 4,4-dimethylfecosterol, and 4-methylfecosterol) and the end product ergosterol are shown.

Figure 2. Low oxygen causes 4-methyl sterol accumulation, ergosterol depletion, and Sre1 activation

Wild-type and sre1Δ yeast were grown in rich medium for the indicated time at 0.5% oxygen. A, Total cell extracts (10 μg) were subjected to immunoblot analysis with anti-Sre1 IgG after treatment with alkaline phosphatase. P and N denote the membrane-bound precursor and cleaved nuclear forms of Sre1, respectively. B–D, Sterols extracted from 5 × 10⁷ cells were analyzed by gas chromatography. Cholesterol (5 μg) was added as an internal standard. Data are the average of three independent replicates. Error bars equal one standard deviation. 4,4-dimethylfecosterol = 4,4-dimethylfecosterol + 4-methylfecosterol in all figures. E, Wild-type yeast containing either empty vector or plasmids expressing Myc-erg11 and HA-erg25 from the nmt promoter were grown in EMM for 20 h prior to growth in rich medium at 21% oxygen (atmospheric) or 0.5% oxygen for 2 h. (upper panel) Total cell extracts (10 μg) were subjected to immunoblot analysis with anti-Sre1 IgG after treatment with alkaline phosphatase. (lower panels) Total cell extracts (40 μg) were subjected to immunoblot analysis with anti-Myc IgG 9E10 or anti-HA-HRP.

Figure 3. Exogenous lanosterol activates Sre1 cleavage

A, Wild-type yeast were grown in rich medium for 2 h in the absence or presence of the indicated concentration of lanosterol. Total cell extracts (10 μg) were subjected to immunoblot analysis with anti-Sre1 IgG after treatment with alkaline phosphatase. P and N denote the membrane-bound precursor and cleaved nuclear forms of Sre1, respectively. The final concentration of ethanol in each culture was 0.6%. B, Wild-type yeast were grown in rich medium for the indicated time in the presence of 60 μg/ml lanosterol. Total cell extracts (10 μg) were subjected to immunoblot analysis with anti-Sre1 IgG after treatment with alkaline phosphatase.

Figure 4. Hydrogen peroxide causes 4-methyl sterol accumulation and Sre1 activation

Wild-type yeast containing either empty vector or plasmids expressing Myc-erg11 and HA-erg25 from the nmt promoter were grown in EMM for 20 h prior to the addition of 500 μM H₂O₂ for the indicated time. A, (upper panel) Total cell extracts (10 μg) were subjected to immunoblot analysis with anti-Sre1 IgG after treatment with alkaline phosphatase. (lower panels) Total cell extracts (40 μg) were subjected to immunoblot analysis with anti-Myc IgG 9E10 or anti-HA-HRP. P and N denote the membrane-bound precursor and cleaved nuclear forms of Sre1, respectively. Asterisk indicates cross reactivity with alkaline phosphatase. B–E, Sterols extracted from 5 × 10⁷ cells were analyzed by gas chromatography. Cholesterol (5 μg) was added as an internal standard. Data are the average of three independent replicates. Error bars equal one standard deviation. (E) 4-methyl sterols = 24-methylene lanosterol + 4-methylfecosterol + 4,4-dimethylfecosterol.

Figure 5. Calcium ionophore A23187 causes 4-methyl sterol accumulation and Sre1 activation

Spheroplasted wild-type yeast were grown in rich medium containing sorbitol for 4 h in the absence or presence of 0.1% DMSO (D) or 1 μM A23187 (A). A, Total cell extracts (10 μg) were subjected to immunoblot analysis with anti-Sre1 IgG after treatment with alkaline phosphatase. P and N denote the membrane-bound precursor and cleaved nuclear forms of Sre1, respectively. B, Sterols extracted from 5 × 10⁷ cells were analyzed by gas chromatography. Cholesterol (5 μg) was added as an internal standard. Data are the average of three independent replicates. Error bars equal one standard deviation.

Figure 6. Exogenous lanosterol activates C. neoformans SREBP

A, C. neoformans cells (strain B3501A) were grown in rich medium for 2 h in the absence or presence of the indicated concentration of lanosterol. Total cell extracts (40 μg) were subjected to immunoblot analysis with anti-CnSre1p IgG. P and N denote the membrane-bound precursor and cleaved nuclear forms of Sre1p, respectively. The final concentration of ethanol in each lane was 0.1%. B, C. neoformans cells were grown in rich medium for the indicated time in the presence of 10 μg/ml lanosterol. Total cell extracts (40 μg) were subjected to immunoblot analysis with anti-CnSre1p IgG.

Figure 7. Sre1 responds to both increased and decreased 4-methyl sterol levels

Wild-type yeast were grown in rich medium for 8 h in the absence or presence of ergosterol synthesis inhibitors: C, 200 μM compactin; I, 100 μM itraconazole; CI, 200 μM compactin plus 100 μM itraconazole. A, Total cell extracts (10 μg) were subjected to immunoblot analysis with anti-Sre1 IgG after treatment with alkaline phosphatase. P and N denote the membrane-bound precursor and cleaved nuclear forms of Sre1, respectively. B, Sterols extracted from 5 × 10⁷ cells were analyzed by gas chromatography. Cholesterol (5 μg) was added as an internal standard. Data are the average of three independent replicates. Error bars equal one standard deviation.

Figure 8. Scp1 SSD is required for sterol sensing and regulates Sre1 activation

A, Alignment of sterol sensing domains from S. pombe Scp1 (residues 244–400) and human Scap (residues 295–451). Identical residues are shaded. Transmembrane segments as predicted by TMHMM 2.0 are underlined and numbered. Asterisks (*) indicate conserved residues that when mutated confer constitutive activity on human Scap (7). B, (upper panel) Total extracts (10 μg) from wild-type yeast carrying an empty vector plasmid or a plasmid overexpressing wild-type Myc-scp1, Myc-scp1 Y247C, Myc-scp1 L264F, or Myc-scp1 D392N from the nmt** promoter were subjected to immunoblot analysis with anti-Sre1 IgG after treatment with alkaline phosphatase. Cells were grown in EMM for 48 h to induce Myc-Scp1 expression. P and N denote the membrane-bound precursor and cleaved nuclear forms of Sre1, respectively. (lower panel) Microsomes (40 μg) were subjected to immunoblot analysis with anti-Myc IgG 9E10.