Altered sterol metabolism in budding yeast affects mitochondrial iron-sulfur (Fe-S) cluster synthesis

Abstract

Ergosterol synthesis is essential for cellular growth and viability of the budding yeast Saccharomyces cerevisiae, and intracellular sterol distribution and homeostasis are therefore highly regulated in this species. Erg25 is an iron-containing C4-methyl sterol oxidase that contributes to the conversion of 4,4-dimethylzymosterol to zymosterol, a precursor of ergosterol. The ERG29 gene encodes an endoplasmic reticulum (ER)-associated protein, and here we identified a role for Erg29 in the methyl sterol oxidase step of ergosterol synthesis. ERG29 deletion resulted in lethality in respiring cells, but respiration-incompetent (Rho^- or Rho⁰) cells survived, suggesting that Erg29 loss leads to accumulation of oxidized sterol metabolites that affect cell viability. Down-regulation of ERG29 expression in Δerg29 cells indeed led to accumulation of methyl sterol metabolites, resulting in increased mitochondrial oxidants and a decreased ability of mitochondria to synthesize iron-sulfur (Fe-S) clusters due to reduced levels of Yfh1, the mammalian frataxin homolog, which is involved in mitochondrial iron metabolism. Using a high-copy genomic library, we identified suppressor genes that permitted growth of Δerg29 cells on respiratory substrates, and these included genes encoding the mitochondrial proteins Yfh1, Mmt1, Mmt2, and Pet20, which reversed all phenotypes associated with loss of ERG29 Of note, loss of Erg25 also resulted in accumulation of methyl sterol metabolites and also increased mitochondrial oxidants and degradation of Yfh1. We propose that accumulation of toxic intermediates of the methyl sterol oxidase reaction increases mitochondrial oxidants, which affect Yfh1 protein stability. These results indicate an interaction between sterols generated by ER proteins and mitochondrial iron metabolism.

Keywords: Fe-S clusters; iron; iron-sulfur protein; mitochondria; sterol; yeast.

Figure 1.

Loss of ERG29 results in loss of the mitochondrial genome and increased levels of 4,4-DMZ and 4-methyl fecosterol. A, Δerg29pβ-estradiolGAL1ERG29 (Rho⁺ or Rho⁰ (spontaneous Rho⁰)) containing empty vector pYCp or pYCpERG29 cells were grown on YPGE or YPD plates in the presence or absence of β-estradiol for 4 days. B, WT (DY1457pβ-estradiolGAL1 vector) and Δerg29pβ-estradiolGAL1ERG29 were grown in galactose or glucose for 16 h, sterols were extracted, and sterol analysis was performed using GC/MS as described under “Materials and methods.” The data are expressed as the percentage of total sterols. Error bars, S.D. n = 3. C, Western blot analysis of Erg29-HIS or Erg25 protein levels in galactose and glucose was performed using His₆ antibody or Erg25 antibody as described under “Materials and methods”. Dpm1, an ER protein, was used as a load control. n = 3. A representative blot is shown. The ratio of Erg29 or Erg25 to Dpm1 was determined using Bio-Rad ImageLab^TM software. D, Δerg29pβ-estradiolGAL1ERG29 containing either empty vector or pYCpERG29 cells was grown in glucose in the absence or presence of β-estradiol for 16 h, and sterol analysis was performed as described in B. The data are expressed as the percentage of total sterols. Error bars, S.D. of four replicates. White bars, no Erg29; white hatched bars, ERG29 expressed under its endogenous promoter; gray bars, +β-estradiol ERG29 expression; gray larger hatched bars, +β-estradiol ERG29 expression and ERG29 expressed under its endogenous promoter. E, WT (DY1457pβ-estradiolGAL1 empty vector) and Δerg25 pGALERG25 cells were grown in galactose- or glucose-containing medium for 16 h as in B, and sterol analysis was performed as described. The Δerg25 samples were prepared, and sterol analysis was done in the same experiment as B to contrast Δerg29 and Δerg25; therefore, the WT data shown is the same in B and E. The data are expressed as the percentage of total sterols. Error bars, S.D. n = 3.

Figure 2.

Erg25 partially suppresses the loss of ER-localized Erg29. A, Δerg29pβ-estradiolGAL1ERG29 transformed with pYEp, pYEpERG25, or pYEpERG26 was grown in galactose or glucose for 3 days. B, Δerg29pβ-estradiolGAL1ERG29 transformed with pMET3YEp or pMET3ERG29-GFP was grown in galactose or glucose minus methionine for 2 days. C, WT cells were transformed with a complementing plasmid containing MET3ERG29-GFP. The cells were grown to mid-log phase in the absence of methionine and examined by epifluorescence microscopy as described under “Materials and methods.” Arrows, typical ER localization surrounding the nucleus and near the plasma membrane similar to Erg25 localization (18). DIC, differential interference contrast.

Figure 3.

Loss of respiration affects the sterol pattern of Δerg29 cells. A, WT Rho⁰ and Δerg29 Rho⁰ cells were grown in CM glucose medium for 18 h, and sterol analysis was performed as described. The data are expressed as the percentage of total sterols. Error bars, S.D. of four separate experiments. B, cells as in A were plated onto iron-limited medium (BPS (0–100 μm iron)) and grown at 30 °C for 2 days. C, sterol analysis was performed on cells grown in iron-limited medium (BPS(0)) for 4 h. The data are expressed as the percentage of total sterols. Error bars, S.D. of two separate experiments. D, cells as in A were plated onto YPD with or without 1 mm CoCl₂ or YPGE and grown at 30 °C for 3 days. E, cells as in A were plated onto CM medium containing varying concentrations (μm) of ABP and grown at 30 °C for 2 days.

Figure 4.

Library screen identifies YFH1, MMT1, MMT2, and PET20 as high-copy suppressors of the loss of ERG29. A, WT cells containing pYEp or Δerg29pGAL1ERG29 cells transformed with pYEp or identified library candidates (YFH1, MMT1, MMT2, or PET20) were plated onto galactose- or glucose-containing medium and grown for 3 days. n = 4. B, sterol analysis was performed on cells as in A, grown in glucose-containing medium for 18 h. The data are expressed as the percentage of total sterols. Error bars, S.D. n = 3.

Figure 5.

Changes in mitochondrial iron levels affect Δerg29 growth on respiratory substrates. A, two separate clones of either Δmrs3Δmrs4 (C1/C2) or Δerg29Δmrs3Δmrs4 (E1/E2) containing a pMET3MRS3 plasmid were grown on GE-containing medium + 10× Met (OFF) or −Met (ON) for 5 days. n = 2. B, Δerg29 Rho⁰ cells containing pMET3, pMET3MRS3, or pMETMRS3 and pMET3MMT1 were grown in the absence of methionine for 8 h, and sterol analysis was performed. The data are expressed as the percentage of total sterols. Error bars, S.D. of two separate experiments.

Figure 6.

Loss of YFH1 or ERG29 affects Fe-S cluster synthesis. A, Δyfh1pMET3YFH1 cells containing empty vector or pMMT1/2 were grown on CM plus (OFF) or minus 10× methionine (ON) with or without 0.00125% H₂O₂ or 3.0 mm iron (Fe) for 3 days. B, WT 1457 and Δerg29 pGAL1ERG29 cells with empty vector, pYFH1, pMMT1, pMMT2, or pPET20 were grown in glucose for 18 h, and aconitase activity was measured as described under “Materials and methods.” n = 3. C, lysates from B were analyzed for lipoic acid protein modification by Western blotting as described previously (35). n = 3. PDH, pyruvate dehydrogenase; KDH, α-ketogluterate dehydrogenase. The ratio of lipoic acid modification/porin was determined using Bio-Rad ImageLab^TM software. Error bars, S.D.

Figure 7.

Loss of ERG29 results in decreased levels of Yfh1 protein and increased mitochondrial oxidants. A, mitochondrial preparations from WTpYEp, Δerg29pGAL1ERG29pYEp, pMMT1, or pPET20 grown in galactose or glucose were analyzed for Yfh1, porin, Nfs1, Isu1, and Ssq1 by Western blotting. n = 2. Gels were quantified using Bio-Rad ImageLab^TM software with Yfh1 levels normalized to porin in each sample. B, mRNA was isolated from WTpYEp or Δerg29 pGAL1ERG29 cells grown in glucose for 16 h, and quantitative PCR was performed for YFH1 or ISU1 and ACT1 for normalization. C, cells as in B were incubated with 20 nm Mitosox dye and washed, and Mitosox fluorescence was determined by flow cytometry in duplicate as described under “Materials and methods.” n = 2 separate experiments. Error bars, S.D.

Figure 8.

Loss of ERG25 results in decreased Fe-S synthesis and decreased Yfh1. A, WT and Δerg25pGAL1ERG25 cells were grown in glucose-containing medium for 16 h, and aconitase activity was measured. n = 2 with two separate clones per experiment. B, mitochondrial lysates from cells as in A grown in galactose or glucose were analyzed for Yfh1, porin, Nfs1, Isu1, and Ssq1by Western blotting. Gels were quantified using Bio-Rad ImageLab^TM software with Yfh1 levels normalized to porin in each sample. C, Δerg25pGAL1ERG25 containing pYEp, pMMT1, pMMT2, or pYFH1 grown in galactose or glucose for 16 h was incubated with 20 nm Mitosox dye and washed, and Mitosox fluorescence was determined by flow cytometry in duplicate as described under “Materials and methods.” n = 2.