Inhibition of sterol synthesis by delta 5-sterols in a sterol auxotroph of yeast defective in oxidosqualene cyclase and cytochrome P-450

Synthesis of ergosterol is demonstrated in the GL7 mutant of Saccharomyces cerevisiae. This sterol auxotroph has been thought to lack the ability to synthesize sterols due both to the absence of 2,3-oxidosqualene cyclase and to a heme deficiency eliminating cytochrome P-450 which is required in demethylation at C-14. However, when the medium sterol was 5 alpha-cholestan-3 beta-ol, 5 alpha-cholest-8(14)-en-3 beta-ol, or 24 beta-methyl-5 alpha-cholest-8(14)-en-3 beta-ol, sterol synthesis was found to proceed yielding 1-3 fg/cell of ergosterol (24 beta-methylcholesta-5,7,22E-trien-3 beta-ol). Ergosterol was identified by mass spectroscopy, gas and high performance liquid chromatography, ultraviolet spectroscopy, and radioactive labeling from [3H]acetate. Except for some cholest-5-en-3 beta-ol (cholesterol) which was derived from the 5 alpha-cholestan-3 beta-ol, the stanol and the two 8(14)-stenols were not significantly metabolized confirming the absence of an isomerase for migration of the double bond from C-8(14) to C-7. Drastic reduction of ergosterol synthesis to not more than 0.06 fg/cell was observed when the medium sterol either had a double bond at C-5, as in the case of cholesterol, or could be metabolized to a sterol with such a bond. Thus, both 5 alpha-cholest-8(9)-en-3 beta-ol and 5 alpha-cholest-7-en-3 beta-ol (lathosterol) were converted to cholesta-5,7-dien-3 beta-ol (7-dehydrocholesterol), and the presence of the latter dienol depressed the level of ergosterol. The most attractive of the possible explanations for our observations is the assumption of two genetic compartments for synthesis of sterols, one of which has and one of which has not been affected by the two mutations. The ability, despite the mutations, to synthesize small amounts of ergosterol which could act to regulate the cell cycle may also explain why this mutant can grow aerobically with cholesterol (acting in the bulk membrane role) as the sole exogenous sterol.     

Effect of allylamine antimycotic agents on fungal sterol biosynthesis measured by sterol side-chain methylation

Sterol side-chain (C-24) methylation was assayed by incorporation of radioactivity from [Me-14C]methionine into the ergosterol fraction in cells of the pathogenic fungi Candida albicans, Candida parapsilosis and Trichophyton mentagrophytes. Methylation at C-24 occurred after nuclear demethylation in all cases. The method was used to measure ergosterol biosynthesis inhibition by the allylamine antimycotics naftifine and SF 86-327, which are known to block squalene epoxidation. In C. albicans cells treated with SF 86-327 (1 mg l-1) to fully inhibit squalene epoxidation, C-24 methylation continued for several hours at about 40% of the control rate. This residual biosynthesis was probably due to methylation of endogenous sterol precursors. The degree of residual biosynthesis in the three fungi correlated well with their susceptibility to SF 86-327. The highly susceptible dermatophyte T. mentagrophytes had negligible residual sterol biosynthesis. These differences were not due to inhibition of methionine uptake. For naftifine (100 mg l-1) there was evidence of a second inhibitory action in C. albicans. A cell-free assay indicated that this was due to direct inhibition of the C-24 methyltransferase.     

A simple method for the isolation of zymosterol from a sterol mutant of Saccharomyces cerevisiae.

A simple method is described for the direct isolation of zymosterol (5 alpha-cholesta-8,24-dien-3 beta-ol) of high purity from a sterol mutant of Saccharomyces cerevisiae. This yeast strain, which is a double mutant of the ERG6 (sterol transmethylase) and ERG2 (C-8 sterol isomerase) genes, accumulates zymosterol as its major sterol component.     

Dissecting the sterol C-4 demethylation process in higher plants. From structures and genes to catalytic mechanism

Sterols become functional only after removal of the two methyl groups at C-4. This review focuses on the sterol C-4 demethylation process in higher plants. An intriguing aspect in the removal of the two C-4 methyl groups of sterol precursors in plants is that it does not occur consecutively as it does in yeast and animals, but is interrupted by several enzymatic steps. Each C-4 demethylation step involves the sequential participation of three individual enzymatic reactions including a sterol methyl oxidase (SMO), a 3β-hydroxysteroid-dehydrogenase/C4-decarboxylase (3βHSD/D) and a 3-ketosteroid reductase (SR). The distant location of the two C-4 demethylations in the sterol pathway requires distinct SMOs with respective substrate specificity. Combination of genetic and molecular enzymological approaches allowed a thorough identification and functional characterization of two distinct families of SMOs genes and two 3βHSD/D genes. For the latter, these studies provided the first molecularly and functionally characterized HSDs from a short chain dehydrogenase/reductase family in plants, and the first data on 3-D molecular interactions of an enzyme of the postoxidosqualene cyclase sterol biosynthetic pathway with its substrate in animals, yeast and higher plants. Characterization of these three new components involved in C-4 demethylation participates to the completion of the molecular inventory of sterol synthesis in higher plants.     

Comparative responses of the yeast mutant strain GL7 to lanosterol,cycloartenol, and cyclolaudenol

Under anaerobic growth conditions the isomeric 4,4′,14-trimethylcholestane derivatives lanosterol and, more efficiently, cycloartenol satisfy the sterol requirement of the yeast sterol auxotroph $\text{Saccharomyces cerevisiae}$ strain GL7. Aerobic mutant growth is supported only by cycloartenol and not by lanosterol, suggesting different structural requirements for aerobic and anaerobic cells. It is proposed that the non-planar conformation imposed by the 9,19-cyclopropane ring of cycloartenol moderates the adverse membrane effects of the nuclear methyl groups at C-4 and C-14. Under both aerobic and anaerobic conditions cyclolaudenol, a C-24-methyl derivative of cycloartenol, is a significantly more effective sterol source for strain GL7 than cycloartenol. This result is in keeping with the predominance of C-24-methyl sterols (ergosterol) in wild-type yeast.     

The ERG28-encoded protein, Erg28p, interacts with both the sterol C-4 demethylation enzyme complex as well as the late biosynthetic protein, the C-24 sterol methyltransferase (Erg6p)

In Saccharomyces cerevisiae, the C-24 sterol methyltransferase (Erg6p) converts zymosterol to fecosterol, an enzymatic step following C-4 demethylation of 4,4-dimethylzymosterol. Our previous study showed that an endoplasmic reticulum (ER) transmembrane protein, Erg28p, functions as a scaffold to tether the C-4 demethylation enzymatic complex (Erg25p-Erg26p-Erg27p) to the ER. To determine whether Erg28p also interacts with other ergosterol biosynthetic proteins, we compared protein levels of Erg3p, Erg6p, Erg7p, Erg11p and Erg25p in three pairs of erg28 and ERG28 strains. In erg28 strains, the Erg6p level in the ER fraction was decreased by about 50% relative to the wild-type strain, while ER protein levels of the four other ergosterol proteins showed no significant differences. Co-immunoprecipitation experiments, using an erg28 strain transformed with the epitope-tagged plasmid pERG28-HA and proteins detected with anti-HA and anti-Erg6p antibodies, indicated that Erg6p and Erg28p reciprocally co-immunoprecipitate. Further, the split ubiquitin yeast membrane two-hybrid system designed to detect protein interactions between membrane bound proteins also indicated an Erg28p-Erg6p interaction when pERG6-Cub was used as the bait and pERG28-NubG was used as the prey. We conclude that Erg28p may not only anchor the C-4 demethylation enzyme complex to the ER but also acts as a protein bridge to the Erg6p enzyme required for the next ergosterol biosynthetic step.     

FadD19 of Rhodococcus rhodochrous DSM43269, a steroid-coenzyme A ligase essential for degradation of C-24 branched sterol side chains

The actinobacterial cholesterol catabolic gene cluster contains a subset of genes that encode β-oxidation enzymes with a putative role in sterol side chain degradation. We investigated the physiological roles of several genes, i.e., fadD17, fadD19, fadE26, fadE27, and ro04690DSM43269, by gene inactivation studies in mutant strain RG32 of Rhodococcus rhodochrous DSM43269. Mutant strain RG32 is devoid of 3-ketosteroid 9α-hydroxylase (KSH) activity and was constructed following the identification, cloning, and sequential inactivation of five kshA gene homologs in strain DSM43269. We show that mutant strain RG32 is fully blocked in steroid ring degradation but capable of selective sterol side chain degradation. Except for RG32ΔfadD19, none of the mutants constructed in RG32 revealed an aberrant phenotype on sterol side chain degradation compared to parent strain RG32. Deletion of fadD19 in strain RG32 completely blocked side chain degradation of C-24 branched sterols but interestingly not that of cholesterol. The additional inactivation of fadD17 in mutant RG32ΔfadD19 also did not affect cholesterol side chain degradation. Heterologously expressed FadD19DSM43269 nevertheless was active toward steroid-C26-oic acid substrates. Our data identified FadD19 as a steroid-coenzyme A (CoA) ligase with an essential in vivo role in the degradation of the side chains of C-24 branched-chain sterols. This paper reports the identification and characterization of a CoA ligase with an in vivo role in sterol side chain degradation. The high similarity (67%) between the FadD19(DSM43269) and FadD19H37Rv enzymes further suggests that FadD19H37Rv has an in vivo role in sterol metabolism of Mycobacterium tuberculosis H37Rv.     

Isolation and characterization of an Arabidopsis thaliana C-8,7 sterol isomerase: functional and structural similarities to mammalian C-8,7 sterol isomerase/emopamil-binding protein

The yeast C-8,7 sterol isomerase contains a polyvalent high-affinity drug binding site similar to mammalian sigma receptors. Exogenously supplied sigma ligands inhibit sterol biosynthesis in yeast, demonstrating a pharmacological relationship between sigma ligand-binding and C-8,7 sterol isomerase activity. We report the isolation of an Arabidopsis thaliana C-8,7 sterol isomerase by functional complementation of the corresponding sterol mutant in yeast and its characterization by exposure to sigma ligands. The yeast erg2 mutant, which lacks the C-8,7 sterol isomerase gene and activity, was transformed with an Arabidopsis cDNA yeast expression library. Transformed colonies were selected for restoration of C-8,7 sterol isomerase activity (i.e. wild-type ergosterol production) by enhanced resistance to the antibiotic cycloheximide. Sterols produced in complemented lines were characterized by gas chromatography-mass spectroscopy (GC-MS). The full-length A. thaliana cDNA (pA.t.SI1) that complemented the erg2 mutation contains an open reading frame encoding a 21 kDa protein that shares 68% similarity and 35% amino acid identity to the recently isolated mouse C-8,7 sterol isomerase. The sigma ligands, haloperidol, ifenprodil and verapamil inhibited the production of ergosterol in wild-type Saccharomyces cerevisiae and in the erg2 mutant complemented with pA.t.SI1. Structural and biochemical similarities between the A. thaliana C-8,7 sterol isomerase and the mammalian emopamil-binding protein (EBP) are discussed.     

Interactions of oxidosqualene cyclase (Erg7p) with 3-keto reductase (Erg27p) and other enzymes of sterol biosynthesis in yeast

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.     

Identification of 24-methylene-24,25-dihydrolanosterol as a precursor of ergosterol in the yeasts Schizosaccharomyces pombe and Schizosaccharomyces octosporus

Abstract Study of the plasma membrane sterol composition in the yeasts Schizosaccharomyces pombe and Schizosaccharomyces octosporus revealed the presence of ergosterol, lanosterol, dehydroergosterol, fecosterol, episterol and 24-methylene-24,25-dihydrolanosterol (eburicol), a C-31 derivative. The growth of both yeasts in the presence of ketoconazole led to a decrease by 85% of the ergosterol content while the levels of lanosterol and eburicol increased. This suggests that in the biosynthetic pathway of ergosterol in Schizosaccharomyces species, the transmethylation process on the C-24 may occur directly on lanosterol and not only on zymosterol. On the other hand, it cannot be excluded that in the genus Schizosaccharomyces two routes exist from lanosterol to ergosterol: the classical one via a direct C-14, C-4 demethylation of lanosterol and the second one via the formation of a C-31 derivative followed by demethylations.     

The biosynthetic incorporation of the intact leucine skeleton into sterol by the trypanosomatid Leishmania mexicana

The amino acid leucine is efficiently used by the trypanosomatid Leishmania mexicana for sterol biosynthesis. The incubation of [2-(13)C]leucine with L. mexicana promastigotes in the presence of ketoconazole gave 14alpha-methylergosta-8,24(24(1))-3beta-ol as the major sterol, which was shown by mass spectrometry to contain up to six atoms of (13)C per molecule. (13)C NMR analysis of the 14alpha-methylergosta-8,24(24(1))-3beta-ol revealed that it was labeled in only six positions: C-2, C-6, C-11, C-12, C-16, and C-23. This established that the leucine skeleton is incorporated intact into the isoprenoid pathway leading to sterol; it is not converted first to acetyl-CoA, as in animals and plants, with utilization of the acetyl-CoA to regenerate 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). An inhibitor of HMG-CoA synthase (L-659,699) blocked the incorporation of [1-(14)C]acetate into sterol but had no inhibitory effect on [U-(14)C]leucine incorporation. The HMG-CoA reductase inhibitor lovastatin inhibited promastigote growth and [U-(14)C]leucine incorporation into sterol. The addition of unlabeled mevalonic acid (MVA) overcame the lovastatin inhibition of growth and also diluted the incorporation of [1-(14)C]leucine into sterol. These results are compatible with two routes by which the leucine skeleton may enter intact into the isoprenoid pathway. The catabolism of leucine could generate HMG-CoA that is then directly reduced to MVA for incorporation into sterol. Alternatively, a compound produced as an intermediate in leucine breakdown to HMG-CoA (e.g. dimethylcrotonyl-CoA) could be directly reduced to produce an isoprene alcohol followed by phosphorylation to enter the isoprenoid pathway post-MVA.     

Effects of ketoconazole on sterol biosynthesis by Trypanosoma cruzi epimastigotes

Reproduction of Trypanosoma cruzi epimastigotes in a liver infusion-tryptose-serum medium was inhibited by the antimycotic agent ketoconazole. Effective drug concentrations also blocked the biosynthesis of the parasite's fungal-type sterols at the stage of C-14 demethylation, as demonstrated by radioisotopic, chromatographic and mass spectrometric methods. Coincidently, a 4,4,14 alpha-trimethyl sterol, 24-methylenedihydrolanosterol, accumulated and came to replace as much as three fourths of the free sterol content of the cells. Precedents from studies with fungi are invoked to suggest that the cytotoxicity of ketoconazole for T. cruzi is a consequence of the inability of 24-methylenedihydrolanosterol to perform the membrane lipid bilayer functions of the normal epimastigote sterols.     

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

A variety of sterols and stanols have been analyzed for their ability to satisfy bulk membrane and high-specificity (sparking) functions in three yeast sterol auxotrophs. While many sterols and stanols satisfied bulk membrane requirements, only those possessing a C-5,6 unsaturation or capable of being desaturated at C-5 fulfilled the high-specificity sparking requirement. Unsaturation of the A-ring or beta-saturation of a C-5,6 double bond rendered both sterol and stanol unsuitable for either function. The C-28 methyl group of ergosterol, while not required for growth, allowed for greater ease of desaturation at C-5 in vivo. As a result some sterols and stanols lacking the C-28 methyl were incapable of satisfying the sparking requirement while identical compounds possessing the C-28 methyl were able to fulfill the sparking function(s). These data are extended to hypothesize a role for the C-28 methyl group of ergosterol in yeast.     

甾醇C-24甲基转移酶和甾醇C-8异构酶在酿酒酵母麦角甾醇生物合成中的调控作用

摘要：【目的】研究ERG6基因编码的甾醇C-24甲基转移酶和ERG2基因编码的甾醇C-8异构酶在酿酒酵母麦角甾醇生物合成代谢中的调控作用。【方法】通过PCR扩增克隆到酿酒酵母甾醇C-8异构酶的编码序列及其终止子序列,以大肠杆菌-酿酒酵母穿梭质粒YEp352为载体,以磷酸甘油酸激酶基因PGK1启动子为上游调控元件构建了酵母菌表达质粒pPERG2;同时,在本实验室已构建的ERG6表达质粒pPERG6的基础上,构建了ERG2和ERG6共表达的重组质粒pPERG6-2。将表达质粒转化酿酒酵母单倍体菌株YS58,依据营养缺陷互补筛选到重组菌株YS58(pPERG2)和YS58(pPERG6-2)。通过紫外分光光度法和气相色谱法分析重组菌株甾醇组分和含量。【结果】在ERG6高表达的重组酵母菌中,甾醇中间体和终产物麦角甾醇的含量均比对照菌高;而在ERG2高表达的酵母菌株中,无论甾醇中间体,还是麦角甾醇的含量均明显降低。ERG6和ERG2共表达重组菌株YS58(pPERG6-2)的麦角甾醇含量是对照菌株YS58(YEp352)的1.41倍,是ERG2单独高表达菌株YS58(pPERG2)的1.92倍,是ERG6单独高表达菌株YS58(pPERG6)的1.12倍。【结论】本研究首次证明甾醇C-24甲基转移酶催化的反应是酿酒酵母麦角甾醇合成代谢途径中的一个重要的限速步骤,该酶活性提高不但补偿了ERG2高表达对甾醇合成的负效应,而且使麦角甾醇含量进一步提高,为构建麦角甾醇高产酵母工程菌株提供了实验依据。     

Cloning, sequencing, and disruption of the gene encoding sterol C-14 reductase in Saccharomyces cerevisiae.

A sterol C-14 reductase (erg24-1) mutant of Saccharomyces cerevisiae was selected in a fen1, fen2, suppressor background on the basis of nystatin resistance and ignosterol (ergosta-8,14-dienol) production. The erg24-1 allele segregated genetically as a single, recessive gene. The wild-type ERG24 gene was cloned by complementation onto a 12-kb fragment from a yeast genomic library, and subsequently subcloned onto a 2.4-kb fragment. This was sequenced and found to contain an open reading frame of 1,314 bp, predicting a polypeptide of 438 amino acids (M(r) 50,612). A 1,088-bp internal region of the ERG24 gene was excised, replaced with a LEU2 gene, and integrated into the chromosome of the parental strain, FP13D (fen1, fen2) by gene replacement. The ERG24 null mutant produced ergosta-8,14-dienol as the major sterol, indicating that the delta 8-7 isomerase, delta 5-desaturase and the delta 22-desaturase were inactive on sterols with the C14 = 15 double bond.     

Mechanistic studies of lanosterol 14 alpha-methyl demethylase: substrate requirements for the component reactions catalyzed by a single cytochrome P-450 isozyme

Lanosterol 14 alpha-methyl demethylation is a cytochrome P-450-dependent process that proceeds through the oxidative sequence of alcohol, aldehyde followed by decarbonylation with formic acid release. Microsomal metabolism studies shown here indicate that only lanostenols and 32-oxy-lanostenols with unsaturation at either the delta 7 or delta 8 position in the sterol can be demethylated. The 14 alpha-methyl group of either lanostan-3 beta-ol or delta 6 lanostenol is not oxidized to the anticipated C-32 alcohol or aldehyde by the enzyme, nor are the corresponding 32-oxy-lanostanols demethylated when incubated with microsomal preparations. Despite the lack of metabolism, the saturated and delta 6 sterol analogues are effective competitive inhibitors of demethylase activity. Utilizing preferred substrates, comparison of the component reactions of the demethylation sequence shows that both the oxidative function and lyase function are sensitive to common inhibitors and that both activities require NADPH. These findings strongly support the premise that a P-450 isozyme does catalyze each phase of the lanosterol 14 alpha-methyl demethylation sequence. Collectively these results demonstrate the double-bond requirement for both components of the demethylation sequence and suggest that the olefinic electrons at delta 7 or delta 8 but not delta 6 may participate directly during demethylation. This participation may involve stabilizing a transition state intermediate or directing activated oxygen insertion as part of the P-450 monoxygenase mechanism.     

Cytochrome P-450-dependent oxidation of lanosterol in cholesterol biosynthesis. Microsomal electron transport and C-32 demethylation

Electron transfer to rat liver microsomal cytochrome P-450 of 14 alpha-methyl group demethylation of 24,25-dihydrolanosterol (C30-sterol) has been studied with a new radio-high-performance liquid chromatography assay. The monooxygenase is dependent upon NADPH plus oxygen, insensitive to CN-, and sensitive to CO. Microsomal oxidation is also sensitive to trypsin digestion, and reactivation is dependent upon the addition of purified, detergent-solubilized cytochrome P-450 reductase. Electron transport of C-32 sterol demethylation can be fully supported by very low concentrations of NADPH (approximately 10 microM) only in the presence of saturating concentrations of NADH (approximately 200 microM) suggesting involvement of cytochrome b5-dependent electron transfer in addition to the NADPH-supported pathway. The cytochrome P-450 of 14 alpha-demethylation has been solubilized with detergents, resolved chromatographically from cytochrome P-450 reductase and cytochrome b5, and fully active C-32 demethylase reconstituted. Incubation of intact microsomes with NADH and very low concentrations of NADPH described above leads to interruption of demethylation without 14 alpha-methyl group elimination. Under these conditions, C-32 oxidation products of the C30-sterol substrate accumulate at the expense of formation of demethylated, C29-sterol products. This enzymic interruption of C-32 demethylation, accumulation of oxygenated C30-sterols, along with subsequent demethylation of the isolated C30-oxysterols under similar oxidative conditions supports the suggestion that 14 alpha-hydroxymethyl and aldehydic sterols are metabolic intermediates of sterol 14 alpha-demethylation. Only very modest inductions of the constitutive cytochrome P-450 isozyme of 14 alpha-methyl sterol oxidase can be obtained with just 2 out of 12 known, potent inducers of mammalian hepatic cytochrome P-450s. Alternatively, administration of complete adjuvant in mineral oil drastically reduces amounts of total microsomal cytochrome P-450 while activity of 14 alpha-methyl sterol oxidase is not affected dramatically. Thus, as much as 2.5-fold enhancement of C-32 oxidase specific activity is obtained when expressed per unit of cytochrome P-450.     

Flux of sterol intermediates in a yeast strain deleted of the lanosterol C-14 demethylase Erg11p

Lanosterol C-14 demethylase Erg11p of the yeast Saccharomyces cerevisiae catalyzes the enzymatic step following formation of lanosterol by the lanosterol synthase Erg7p in lipid particles (LP). Localization experiments employing microscopic inspection and cell fractionation revealed that Erg11p in contrast to Erg7p is associated with the endoplasmic reticulum (ER). An erg11Delta mutation in erg3Delta background, which is required to circumvent lethality of the erg11 defect, did not only change the sterol pattern but also the sterol distribution within the cell. Whereas in wild type the plasma membrane was highly enriched in ergosterol and LP harbored large amounts of sterol precursors in the form of steryl esters, sterol intermediates were more or less evenly distributed among organelles of erg11Delta erg3Delta. This distribution is not result of the erg3Delta background, because in the erg3Delta strain the major intermediate formed, ergosta-7,22-dienol, is also highly enriched in the plasma membrane similar to ergosterol in wild type. These results indicate that (i) exit of lanosterol from LP occurs independently of functional Erg11p, (ii) random supply of sterol intermediates to all organelles of erg11Delta erg3Delta appears to compensate for the lack of ergosterol in this mutant, and (iii) preferential sorting of ergosterol in wild type, but also of ergosta-7,22-dienol in erg3Delta, supplies sterol to the plasma membrane.