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Sterols are essential lipids, involved in many biological processes. In Saccharomyces cerevisiae, the enzymes of the ergosterol biosynthetic pathway (Erg proteins) are localized in different cellular compartments. With the aim of studying organelle interactions, we discovered that Erg27p resides mainly in Lipid Droplets (LDs) in respiratory competent cells, while in absence of respiration, is found mostly in the ER. The results presented in this paper demonstrate an interplay between the mitochondrial respiration and ergosterol production: on the one hand, rho° cells show lower ergosterol content when compared with wild type respiratory competent cells, on the other hand, the ergosterol biosynthetic pathway influences the mitochondrial status, since treatment with ketoconazole, which blocks the ergosterol pathway, or the absence of the ERG27 gene, induced rho° production in S. cerevisiae. The loss of mitochondrial DNA in the ∆erg27 strain is fully suppressed by exogenous addition of ergosterol. These data suggest the notion that ergosterol is essential for maintaining the mitochondrial DNA attached to the inner mitochondrial membrane., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A comparative study on post-squalene sterol synthesis in intact yeast cells and spheroplasts was carried out with strains from three genera (Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris) as well as with engineered S. cerevisiae cells altered in regard to the late ergosterol synthesis pathway. A common outcome of incubation experiments with radioactive acetate was that in intact cells the metabolic pathway flows till its specific end product (ergosterol and its precursor, depending on the enzyme deficiency), whereas in spheroplasts the pathway was stalled some step upstream. For example, in spheroplasts from wt strains, non-cyclic triterpenes squalene and oxidosqualene accumulated as though the metabolic path was kept from producing steroid-shaped molecules different from the end product. Accumulation of non-cyclic triterpenes was observed also in spheroplasts from S. cerevisiae cells lacking 3-ketosteroid reductase activity, an enzyme belonging to the C4-demethylase complex. When production of cyclic triterpenes was compromised by loss or poor functionality of oxidosqualene cyclase (EC 5.4.99.7), the difference between intact cells and spheroplasts was still remarkable, yet limited to the different oxido/dioxidosqualene ratio. The characteristics of spheroplasts as non-proliferating cells may partially explain the observed differences in post-squalene pathway from intact cells. We cannot say if the difference in metabolic pathways in spheroplasts and intact cells is a rule. We think, however, that it is worthwhile to search for an answer, as a wider picture of the points where the metabolic pathways are stalled in spheroplasts could provide original ideas about the metabolic network in yeast.

Abstract: In Saccharomyces cerevisiae and Candida albicans, two enzymes of the ergosterol biosynthetic pathway, oxidosqualene cyclase (Erg7p) and 3-keto reductase (Erg27p) interact such that loss of the 3-keto reductase also results in a concomitant loss of activity of the upstream oxidosqualene cyclase. This interaction wherein Erg27p has a stabilizing effect on Erg7p was examined to determine whether Erg7p reciprocally has a protective effect on Erg27p. To this aim, three yeast strains each lacking the ERG7 gene were tested for 3-ketoreductase activity by incubating either cells or cell homogenates with unlabeled and radiolabeled 3-ketosteroids. In these experiments, the ketone substrates were effectively reduced to the corresponding alcohols, providing definitive evidence that oxidosqualene cyclase is not required for the 3-ketoreductase activity. This suggests that, in S. cerevisiae, the protective relationship between the 3-keto reductase (Erg27p) and oxidosqualene cyclase (Erg7p) is not reciprocal. However, the absence of the Erg7p, appears to affect other enzymes of sterol biosynthesis downstream of lanosterol formation. Following incubation with radiolabeled and non-radiolabeled 3-ketosteroids we detected differences in hydroxysteroid accumulation and ergosterol production between wild-type and ERG7 mutant strains. We suggest that oxidosqualene cyclase affects Erg25p (C-4 sterol oxidase) and/or Erg26p (C-3 sterol dehydrogenase/C-4 decarboxylase), two enzymes that, in conjunction with Erg27p, are involved in C-4 sterol demethylation. [Copyright &y& Elsevier]