Cloning, disruption and sequence of the gene encoding yeast C-5 sterol desaturase

doi:10.1016/0378-1119(91)90535-J

Gene

Volume 102, Issue 1, 15 June 1991, Pages 39-44

https://doi.org/10.1016/0378-1119(91)90535-J Get rights and content

Abstract

The ERG3 gene from Saccharomyces cerevisiae has been cloned by complementation of an erg3-2 mutation. ERG3 is the putative gene encoding the C-5 sterol desaturase required for ergosterol biosynthesis. The functional gene has been localized on a 2.5-kb HindIII-BamHI fragment containing an open reading frame comprising 365 amino acids. Gene disruption resulting from a deletion/substitution demonstrates that ERG3 is not essential for cell viability or the sparking function.

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Long noncoding RNA lincsc5d regulates hepatic cholesterol synthesis by modulating sterol C5 desaturase in large yellow croaker
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Although long noncoding RNA (lncRNA) plays a vital role in cholesterol metabolism, very little information is available in fish. Thus, a 10-week feeding experiment was performed to estimate the effects of lncRNA on cholesterol metabolism in large yellow croaker fed with fish oil (FO), soybean oil (SO), olive oil (OO), and palm oil (PO) diets. Results showed that fish fed with OO and PO diets had higher liver total cholesterol (TC) and cholesterol ester (CE) contents compared with fish fed with FO diets. Analysis of the KEGG pathway showed that the steroid biosynthesis pathway was enriched in comparisons FO vs SO, FO vs OO, and FO vs PO. Meanwhile, sterol C5 desaturase (SC5D), a cholesterol synthase, was up-regulated in the steroid biosynthesis pathway. SC5D was widely expressed in all tissues examined, and the highest expression of SC5D was detected in brain. More importantly, a novel lncRNA associated with sc5d gene was identified by RNA sequencing and named as lincsc5d. The tissue distribution of lincsc5d was similar to that of sc5d. A nuclear/cytoplasmic RNA separation assay showed that lincsc5d was a nucleus-enriched lncRNA. qRT-PCR results demonstrated that lincsc5d was markedly up-regulated in the SO, OO, and PO groups. Furthermore, the results of TC content and the lincsc5d and sc5d expression in hepatocytes agreed with in vivo results. In conclusion, this study indicated that vegetable oils, especially OO and PO, increased hepatic cholesterol levels by promoting cholesterol synthesis, and lncRNA lincsc5d and sc5d might be involved in cholesterol synthesis.
A novel Tetrahymena thermophila sterol C-22 desaturase belongs to the fatty acid hydroxylase/desaturase superfamily
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Citation Excerpt :
These results, together with the parametric analysis, indicate a gene duplication which gave rise to a functional divergence as the most plausible scenario for the origin of the C-22 desaturase. To date, three sterol desaturases that belong to different EC nodes have been identified: C-5, C-7 and C-22 desaturases (23, 34, 35). Although the three enzymes are integral membrane monooxygenases with apparent similarity to the reaction that they catalyze, the three enzymes do not share phylogenetic patterns, functional domains, topological motifs, nor electron transfer chains (ETCs).
Sterols in eukaryotic cells play important roles in modulating membrane fluidity and in cell signaling and trafficking. During evolution, a combination of gene losses and acquisitions gave rise to an extraordinary diversity of sterols in different organisms. The sterol C-22 desaturase identified in plants and fungi as a cytochrome P-450 monooxygenase evolved from the first eukaryotic cytochrome P450 and was lost in many lineages. Although the ciliate Tetrahymena thermophila desaturates sterols at the C-22 position, no cytochrome P-450 orthologs are present in the genome. Here, we aim to identify the genes responsible for the desaturation as well as their probable origin. We used gene knockout and yeast heterologous expression approaches to identify two putative genes, retrieved from a previous transcriptomic analysis, as sterol C-22 desaturases. Furthermore, we demonstrate using bioinformatics and evolutionary analyses that both genes encode a novel type of sterol C-22 desaturase that belongs to the large fatty acid hydroxylase/desaturase superfamily and the genes originated by genetic duplication prior to functional diversification. These results stress the widespread existence of nonhomologous isofunctional enzymes among different lineages of the tree of life as well as the suitability for the use of T. thermophila as a valuable model to investigate the evolutionary process of large enzyme families.
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Citation Excerpt :
For instance, each of the endogenous Hrd1 targets in the T-MAP is involved in sterol metabolism. Erg3, Erg5, and Erg25 are enzymes of ergosterol de novo synthesis (Arthington et al., 1991; Jaenicke et al., 2011; Kelly et al., 1995; Li and Kaplan, 1996), YJR015W encodes a protein with strong homology to human ABC transporters mediating the cellular efflux of sterols (HHPred; Figure S4A), and Ysp2/Ltc4 belongs to the recently discovered family of StART-domain proteins that localize at membrane contact sites and are responsible for the transport of sterols (Gatta et al., 2015; Murley et al., 2015). These findings expand our understanding of Hrd1-dependent ERAD, not only as a master regulator of sterol de novo synthesis but also sterol transport in cells.
Protein degradation is mediated by an expansive and complex network of protein modification and degradation enzymes. Matching degradation enzymes with their targets and determining globally which proteins are degraded by the proteasome or lysosome/vacuole have been a major challenge. Furthermore, an integrated view of protein degradation for cellular pathways has been lacking. Here, we present an analytical platform that combines systematic gene deletions with quantitative measures of protein turnover to deconvolve protein degradation pathways for Saccharomyces cerevisiae. The resulting turnover map (T-MAP) reveals target candidates of nearly all E2 and E3 ubiquitin ligases and identifies the primary degradation routes for most proteins. We further mined this T-MAP to identify new substrates of ER-associated degradation (ERAD) involved in sterol biosynthesis and to uncover regulatory nodes for sphingolipid biosynthesis. The T-MAP approach should be broadly applicable to the study of other cellular processes, including mammalian systems.
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Triterpenoids are the most numerous and structurally diverse natural products, which are widely distributed in bacteria, fungi, plants, and animals. They possess a wide range of biological functions involved in cell membrane construction, signal transduction, protection against pathogens, and so on. Despite the large chemical diversity of triterpenes, they are formed from two linear isoprenoid precursors squalene and oxidosqualene. The huge diversity of triterpenes is attributed to the variety of triterpene synthases and tailoring enzymes. Here, we summarize recent progress in the field of triterpene biosynthesis and give an overview of triterpene biosynthetic pathways, genes and enzymes.
TORC1 ensures membrane trafficking of Tat2 tryptophan permease via a novel transcriptional activator Vhr2 in budding yeast
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Citation Excerpt :
Thus, ergosterol biogenesis is important for maintenance of lipid raft structures and proper membrane trafficking. The yeast ERG genes encoding enzymes involved in the later steps of ergosterol biosynthesis (ERG6, ERG2, ERG3, ERG5, and ERG4) (Fig. 1) are not essential for cell viability [13–16]. However, these erg strains show synthetic lethality with mutations of TRP1 (encoding an enzyme for tryptophan synthesis), which is alleviated by the addition of high levels of tryptophan [9,17].
The target of rapamycin complex 1 (TORC1) protein kinase is activated by nutrients and controls nutrient uptake via the membrane trafficking of various nutrient permeases. However, its molecular mechanisms remain elusive. Cholesterol (ergosterol in yeast) in conjunction with sphingolipids forms tight-packing microdomains, “lipid rafts”, which are critical for intracellular protein sorting. Here we show that a novel target of rapamycin (TOR)-interacting transcriptional activator Vhr2 is required for full expression of some ERG genes for ergosterol biogenesis and for proper sorting of the tryptophan permease Tat2 in budding yeast. Loss of Vhr2 caused sterol biogenesis disturbance and Tat2 missorting. TORC1 activity maintained VHR2 transcript and protein levels, and total sterol levels. Vhr2 was not involved in regulation of the TORC1-downstream protein kinase Npr1, which regulates Tat2 sorting. This study suggests that TORC1 regulates nutrient uptake via sterol biogenesis.

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Gene

Abstract

References (20)

Inhibition of ergosterol biosynthesis Saccharomyces cerevisiae and Ustilago maydis by tridemorph, fenpropimorph and fenpropidin

Phytochemistry

A simple method for displaying the hydropathic character of a protein

J. Mol. Biol.

Inhibition of sterol synthesis by Δ⁵-sterols in a sterol auxotroph of yeast defective in oxidosqualene cyclase and cytochrome P-450

J. Biol. Chem.

Stereochemical specificity for sterols in Saccharomyces cerevisiae

J. Biol. Chem.

Structural and physiological features of sterols necessary to satisfy bulk membrane and sparking sterol requirements in yeast auxotrophs

Arch. Biochem. Biophys.

Saccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector

Gene

Cloning and disruption of the yeast C-8 sterol isomerase gene

Lipids

Procedures for linker insertion mutagenesis and use of new kanamycin resistance casettes

DNA Prot. Eng. Techn.

Biochemical and genetic aspects of nystatin resistance in Saccharomyces cerevisiae

J. Bacteriol.

Biosynthesis of terpenes and steroids, IX. The sterols of some mutant yeasts and their relationship to the biosynthesis of ergosterol

J. Chem. Soc. Perkin Trans.

Cited by (126)

Resistance mechanisms of Saccharomyces cerevisiae against silver nanoparticles with different sizes and coatings

Long noncoding RNA lincsc5d regulates hepatic cholesterol synthesis by modulating sterol C5 desaturase in large yellow croaker

A novel Tetrahymena thermophila sterol C-22 desaturase belongs to the fatty acid hydroxylase/desaturase superfamily

A Systematic Protein Turnover Map for Decoding Protein Degradation

Biosynthesis of triterpenoid natural products

TORC1 ensures membrane trafficking of Tat2 tryptophan permease via a novel transcriptional activator Vhr2 in budding yeast