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Effects of Culture Conditions on Ergosterol Biosynthesis …

Exogenous ergosterol triggered nearly all of the genes that encode the biosynthesis of ergosterol.

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Aspects of ergosterol biosynthesis in Saccharomyces cerevisiae

N2 - The ERGS gene from Saccharomyces cerevisiae was cloned by complementation of an erg5-1 mutation using a negative selection protocol involving screening for nystatin-sensitive transformants. ERG5 is the putative gene encoding the C-22 sterol desaturase required in ergosterol biosynthesis. The functional gene was localized to a 2.15-kb SacI-EcoRI DNA fragment containing an open reading frame of 538 amino acids (aa). ERG5 contains a 10-aa motif consistent with its role as a cytochrome P-450 (CyP450) enzyme and is similar to a number of mammalian CyP450 enzymes. Gene disruption demonstrates that ERG5 is not essential for cell viability.

Effects of Culture Conditions on Ergosterol Biosynthesis by Saccharomyces cerevisiae

AB - Ergosterol is an important constituent of fungal membranes. Azoles inhibit ergosterol biosynthesis, although the cellular basis for their antifungal activity is not understood. We used multiple approaches to demonstrate a critical requirement for ergosterol in vacuolar H+-ATPase function, which is known to be essential for fungal virulence. Ergosterol biosynthesis mutants of S. cerevisiae failed to acidify the vacuole and exhibited multiple vma~ phenotypes. Extraction of ergosterol from vacuolar membranes also inactivated V-ATPase without disrupting membrane association of its subdomains. In both S. cerevisiae and the fungal pathogen C. albicans, fluconazole impaired vacuolar acidification, whereas concomitant ergosterol feeding restored V-ATPase function and cell growth. Furthermore, fluconazole exacerbated cytosolic Ca2+ and H+ surges triggered by the antimicrobial agent amiodarone, and impaired Ca2+ sequestration in purified vacuolar vesicles. These findings provide a mechanistic basis for the synergy between azoles and amiodarone observed in vitro. Moreover, we show the clinical potential of this synergy in treatment of systemic fungal infections using a murine model of Candidiasis. In summary, we demonstrate a new regulatory component in fungal V-ATPase function, a novel role for ergosterol in vacuolar ion homeostasis, a plausible cellular mechanism for azole toxicity in fungi, and preliminary in vivo evidence for synergism between two antifungal agents. New insights into the cellular basis of azole toxicity in fungi may broaden therapeutic regimens for patient populations afflicted with systemic fungal infections.

Saccharomyces cerevisiae S288c Ergosterol Biosynthesis

AB - The ERGS gene from Saccharomyces cerevisiae was cloned by complementation of an erg5-1 mutation using a negative selection protocol involving screening for nystatin-sensitive transformants. ERG5 is the putative gene encoding the C-22 sterol desaturase required in ergosterol biosynthesis. The functional gene was localized to a 2.15-kb SacI-EcoRI DNA fragment containing an open reading frame of 538 amino acids (aa). ERG5 contains a 10-aa motif consistent with its role as a cytochrome P-450 (CyP450) enzyme and is similar to a number of mammalian CyP450 enzymes. Gene disruption demonstrates that ERG5 is not essential for cell viability.

AB - The sterols of six double mutants of Saccharomyces cerevisiae have been isolated and characterised, and the sterol distribution has been related to the biosynthetic pathway from zymosterol to ergosterol. Hitherto unknown ergosta-5,8-dien-3β-ol and cholesta-7,24-dien-3β-ol have been isolated and characterised.

Protein trafficking, ergosterol biosynthesis and membrane …

N2 - The sterols of six double mutants of Saccharomyces cerevisiae have been isolated and characterised, and the sterol distribution has been related to the biosynthetic pathway from zymosterol to ergosterol. Hitherto unknown ergosta-5,8-dien-3β-ol and cholesta-7,24-dien-3β-ol have been isolated and characterised.

N2 - Ergosterol is an important constituent of fungal membranes. Azoles inhibit ergosterol biosynthesis, although the cellular basis for their antifungal activity is not understood. We used multiple approaches to demonstrate a critical requirement for ergosterol in vacuolar H+-ATPase function, which is known to be essential for fungal virulence. Ergosterol biosynthesis mutants of S. cerevisiae failed to acidify the vacuole and exhibited multiple vma~ phenotypes. Extraction of ergosterol from vacuolar membranes also inactivated V-ATPase without disrupting membrane association of its subdomains. In both S. cerevisiae and the fungal pathogen C. albicans, fluconazole impaired vacuolar acidification, whereas concomitant ergosterol feeding restored V-ATPase function and cell growth. Furthermore, fluconazole exacerbated cytosolic Ca2+ and H+ surges triggered by the antimicrobial agent amiodarone, and impaired Ca2+ sequestration in purified vacuolar vesicles. These findings provide a mechanistic basis for the synergy between azoles and amiodarone observed in vitro. Moreover, we show the clinical potential of this synergy in treatment of systemic fungal infections using a murine model of Candidiasis. In summary, we demonstrate a new regulatory component in fungal V-ATPase function, a novel role for ergosterol in vacuolar ion homeostasis, a plausible cellular mechanism for azole toxicity in fungi, and preliminary in vivo evidence for synergism between two antifungal agents. New insights into the cellular basis of azole toxicity in fungi may broaden therapeutic regimens for patient populations afflicted with systemic fungal infections.

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  • Ergosterol biosynthesis in novel melanized fungi from …

    Saccharomyces cerevisiae S288c superpathway of ergosterol biosynthesis

  • Ergosterol biosynthesis pathway in Aspergillus fumigatus

    Summary: This class contains pathways used for the biosynthesis of ergosterol. Parent Classes: Sterol Biosynthesis

  • Physiological Implications of Sterol Biosynthesis in …

    The ergosterol biosynthesis pathway is a complex route in which about 20 enzymes are involved

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Physiological Implications of Sterol Biosynthesis in Yeast.

The sterols of six double mutants of Saccharomyces cerevisiae have been isolated and characterised, and the sterol distribution has been related to the biosynthetic pathway from zymosterol to ergosterol. Hitherto unknown ergosta-5,8-dien-3β-ol and cholesta-7,24-dien-3β-ol have been isolated and characterised.

Aspects of ergosterol biosynthesis in Saccharomyces ..

The two fungal enzymes, C-14 sterol reductase (gene ERG24 in budding yeast and erg3 in Neurospora crassa) and C-24(28) sterol reductase (gene ERG4 in budding yeast and sts1 in fission yeast), are involved in ergosterol biosynthesis. They act by reducing double bonds in precursors of ergosterol []. These proteins are highly hydrophobic and seem to contain seven or eight transmembrane regions. Chicken lamin B receptor that is thought to anchor the lamina to the inner nuclear membrane belongs to this family.

The ergosterol biosynthesis pathway is required for ..

The ERGS gene from Saccharomyces cerevisiae was cloned by complementation of an erg5-1 mutation using a negative selection protocol involving screening for nystatin-sensitive transformants. ERG5 is the putative gene encoding the C-22 sterol desaturase required in ergosterol biosynthesis. The functional gene was localized to a 2.15-kb SacI-EcoRI DNA fragment containing an open reading frame of 538 amino acids (aa). ERG5 contains a 10-aa motif consistent with its role as a cytochrome P-450 (CyP450) enzyme and is similar to a number of mammalian CyP450 enzymes. Gene disruption demonstrates that ERG5 is not essential for cell viability.

Ergosterol biosynthesis: a fungal pathway for life on …

Ergosterol is an important constituent of fungal membranes. Azoles inhibit ergosterol biosynthesis, although the cellular basis for their antifungal activity is not understood. We used multiple approaches to demonstrate a critical requirement for ergosterol in vacuolar H+-ATPase function, which is known to be essential for fungal virulence. Ergosterol biosynthesis mutants of S. cerevisiae failed to acidify the vacuole and exhibited multiple vma~ phenotypes. Extraction of ergosterol from vacuolar membranes also inactivated V-ATPase without disrupting membrane association of its subdomains. In both S. cerevisiae and the fungal pathogen C. albicans, fluconazole impaired vacuolar acidification, whereas concomitant ergosterol feeding restored V-ATPase function and cell growth. Furthermore, fluconazole exacerbated cytosolic Ca2+ and H+ surges triggered by the antimicrobial agent amiodarone, and impaired Ca2+ sequestration in purified vacuolar vesicles. These findings provide a mechanistic basis for the synergy between azoles and amiodarone observed in vitro. Moreover, we show the clinical potential of this synergy in treatment of systemic fungal infections using a murine model of Candidiasis. In summary, we demonstrate a new regulatory component in fungal V-ATPase function, a novel role for ergosterol in vacuolar ion homeostasis, a plausible cellular mechanism for azole toxicity in fungi, and preliminary in vivo evidence for synergism between two antifungal agents. New insights into the cellular basis of azole toxicity in fungi may broaden therapeutic regimens for patient populations afflicted with systemic fungal infections.

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