Cloning and characterization of the Saccharomyces cerevisiae C-22 sterol desaturase gene, encoding a second cytochrome P-450 involved in ergosterol biosynthesis

Skaggs, Benjamin; F.Alexander, John; Pierson, C. A.; Schweitzer, Kelly S.; Chun, Kristin T.; Koegel, C.; Barbuch, Robert J.; Bard, M.

doi:10.1016/0378-1119(95)00770-9

Cited by 109 publications

(82 citation statements)

References 18 publications

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“…A fungal cytochrome P450 monooxygenase, CYP61, is known as the sterol C-22 desaturase to produce ergosta-5,7,22,24(28)-tetraenol from the immediate precursor, ergosta-5,7,24(28)-trienol, at the penultimate step in the ergosterol biosynthetic pathway (Skaggs et al, 1996;Kelly et al, 1997). A sequence comparison (Figure 2) revealed striking sequence conservation as [FLFA(A/S)QDAS(T/S)S] between plant CYP710A proteins and fungal CYP61 proteins, while the overall sequence similarity is only ;30%.…”

Section: Introductionmentioning

confidence: 99%

Cytochrome P450CYP710AEncodes the Sterol C-22 Desaturase inArabidopsisand Tomato

et al. 2006

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D22-Unsaturated sterols, containing a double bond at the C-22 position in the side chain, occur specifically in fungi and plants. Here, we describe the identification and characterization of cytochrome P450s belonging to the CYP710A family as the plant C-22 desaturase. Recombinant proteins of CYP710A1 and CYP710A2 from Arabidopsis thaliana and CYP710A11 from tomato (Lycopersicon esculentum) were expressed using a baculovirus/insect system. The Arabidopsis CYP710A1 and tomato CYP710A11 proteins exhibited C-22 desaturase activity with b-sitosterol to produce stigmasterol (CYP710A1, K m ¼ 1.0 mM and kinetic constant [k cat ] ¼ 0.53 min ÿ1 ; CYP710A11, K m ¼ 3.7 mM and k cat ¼ 10 min ÿ1 ). In Arabidopsis transgenic lines with CYP710A1 and CYP710A11 overexpression, stigmasterol levels increased by 6-to 32-fold. Arabidopsis CYP710A2 was able to produce brassicasterol and stigmasterol from 24-epi-campesterol and b-sitosterol, respectively. Sterol profiling analyses for CYP710A2 overexpression and a T-DNA insertion event into CYP710A2 clearly demonstrated in planta that CYP710A2 was responsible for both brassicasterol and stigmasterol production. Semiquantitative PCR analyses and promoter:b-glucuronidase transgenic approaches indicated strict tissue/organ-specific regulation for each CYP710A gene, implicating differential tissue distributions of the D 22 -unsaturated sterols in Arabidopsis. Our results support the possibility that the CYP710 family may encode P450s of sterol C-22 desaturases in different organisms.

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Section: Introductionmentioning

confidence: 99%

Cytochrome P450CYP710AEncodes the Sterol C-22 Desaturase inArabidopsisand Tomato

et al. 2006

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“…Erg25p [36], Erg11p [37], Erg5p [38] and Erg3p [39]. Therefore, heme deficiency can be detected indirectly by analysis of sterol composition [15,31].…”

Section: Sut1 Expression Results Into a Heme-independent Sterol Uptakmentioning

confidence: 99%

SUT1 is a putative Zn[II]2Cys6‐transcription factor whose upregulation enhances both sterol uptake and synthesis in aerobically growing Saccharomyces cerevisiae cells

Ness

Bourot

Régnacq

et al. 2001

European Journal of Biochemistry

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Budding yeast Saccharomyces cerevisiae is a facultative anaerobe whose growth upon oxygen starvation depends on its capacity to import exogenously supplied sterols, whereas the cells are not permeable to these molecules when grown aerobically. Few genes have been identified as being involved in sterol uptake. A higher SUT1 gene dosage leads to a modest, but significant, increase in sterol uptake under aerobic conditions. Based on sequence and physiological data, SUT1 is a hypoxic gene negatively regulated when the cells are grown in the presence of oxygen. We replaced the SUT1 promoter with the constitutive PMA1 gene promoter in order to enhance its transcription. We observed that sterol uptake was then comparable with that obtained with a sterol importing hem1 mutant, although the heme status of the strain was not modified in a process which still occurs when the cells are not growing. Unexpectedly, SUT1 constitutive expression led to a parallel significant increase in endogenous sterol biosynthesis. Moreover, here we present new data showing that the structurally related YPR009 gene (SUT2) is a functional homologue of SUT1, and that both gene products may represent two novel yeast regulatory proteins involved in sterol uptake.

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“…*, p Ͻ 0.001; **, p Ͻ 0.01. rate-limiting step, the conversion of hydroxymethylglutaryl-CoA to mevalonic acid, and ⌬hmg1 cells exhibit largely reduced enzyme activity (35). Erg5p is the enzyme that desaturates C-22 of the side chain in the late step of ergosterol synthesis (36). Hem14p converts protoporphyrinogen IX to protoporphyrin IX in the late step of heme synthesis (28,29), so in ⌬hem14 cells, the function of heme-containing proteins such as the ergosterol synthesis-related enzymes Erg5p and Erg11p (36,37) would be reduced.…”

Section: Discussionmentioning

confidence: 99%

“…Erg5p is the enzyme that desaturates C-22 of the side chain in the late step of ergosterol synthesis (36). Hem14p converts protoporphyrinogen IX to protoporphyrin IX in the late step of heme synthesis (28,29), so in ⌬hem14 cells, the function of heme-containing proteins such as the ergosterol synthesis-related enzymes Erg5p and Erg11p (36,37) would be reduced. Moreover, the expression of HMG1 and ERG11, regulated by heme at the transcriptional level (32,33), would be also decreased.…”

Section: Discussionmentioning

confidence: 99%

Regulation of the Sphingoid Long-chain Base Kinase Lcb4p by Ergosterol and Heme

Sano

Kihara

Kurotsu

et al. 2005

Journal of Biological Chemistry

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Sphingoid long-chain base 1-phosphates (LCBPs) are widely conserved, bioactive lipid molecules. In yeast, LCBPs are known to be involved in several cellular responses such as heat shock resistance and Ca 2؉ mobilization, although their target molecules and signaling pathways remain unclear. To identify genes involved in LCBP signaling and in regulation of LCBP synthesis, we performed transposon mutagenesis in cells lacking the LCBP lyase DPL1 and LCBP phosphatase LCB3 genes and screened for phytosphingosine-resistant clones. Further isolation and identification revealed eight genes (PBP1, HEM14, UFD4, HMG1, TPS1, KES1, WHI2, and ERG5), in addition to the previously characterized LCB4 and PDR5 genes, that are involved in phytosphingosine resistance. Of these eight, four are ergosterol-related genes (HEM14, HMG1, KES1, and ERG5). We also demonstrated that protein expression of the long-chain base kinase Lcb4p was reduced in ⌬hem14 and ⌬hmg1 cells, likely as a consequence of decreased activity of the heme-dependent transcription factor Hap1p. In addition, phosphorylation of Lcb4p was decreased in all the ergosterol-related mutants isolated and other ergosterol mutants constructed (⌬erg2, ⌬erg3, and ⌬erg6). Finally, plasma membrane localization of Lcb4p was found to be reduced in ⌬erg6 cells. These results suggest that changes in sterol composition affect the phosphorylation of Lcb4p because of the altered localization. The other genes isolated (PBP1, UFD4, TPS1, and WHI2) may be involved in LCBP signaling.Sphingolipids are major membrane components of eukaryotic cells. Ceramide, the backbone of sphingolipids, is formed by the amide linkage of a sphingoid long-chain base (LCB) 2 with a fatty acid. The major mammalian LCB is sphingosine, whereas in the yeast Saccharomyces cerevisiae, dihydrosphingosine (DHS) and phytosphingosine (PHS) serve as LCBs. LCBs are bioactive lipid molecules involved in apoptosis, endocytosis, and G 1 cell cycle arrest (1-3). The phosphorylation of LCBs at C-1 results in the production of long-chain base 1-phosphates (LCBPs), sphingosine 1-phosphate (S1P) in mammals and PHS 1-phosphate (PHS1P) and DHS 1-phosphate in yeast. Interestingly, LCBPs possess completely different biological functions compared with the original LCBs.In mammalian cells, S1P is known to elicit various cellular responses via the cell-surface SIP/Edg (endothelial differentiation gene) receptors, such as proliferation, motility, differentiation, and immunity (4 -7). In addition to its role as an extracellular signal mediator, S1P also functions intracellularly. Intracellular S1P has been proposed to be involved in Ca 2ϩ mobilization, cell proliferation, G 1 /S cell cycle transition, and inhibition of apoptosis (4 -7), although its intracellular target molecules and signaling pathways remain unclear.Because yeast cells do not possess a cell-surface receptor for LCBP/ S1P, their LCBPs function only intracellularly (8 -11). Yeast LCBPs and mammalian intracellular S1P share some effects. For instance, both influence Ca 2ϩ mob...

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Cloning and characterization of the Saccharomyces cerevisiae C-22 sterol desaturase gene, encoding a second cytochrome P-450 involved in ergosterol biosynthesis

Cited by 109 publications

References 18 publications

Cytochrome P450CYP710AEncodes the Sterol C-22 Desaturase inArabidopsisand Tomato

Cytochrome P450CYP710AEncodes the Sterol C-22 Desaturase inArabidopsisand Tomato

SUT1 is a putative Zn[II]2Cys6‐transcription factor whose upregulation enhances both sterol uptake and synthesis in aerobically growing Saccharomyces cerevisiae cells

Regulation of the Sphingoid Long-chain Base Kinase Lcb4p by Ergosterol and Heme

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