The effect of altered sterol composition on cytochrome oxidase and S-adenosylmethionine: delta 24 sterol methyltransferase enzymes of yeast mitochondria

Arrhenius kinetics of two mitochondrial enzymes, cytochrome oxidase and S-adenosylmethionine: Δ 24 sterol methyltransferase were analyzed in wild-type and sterol mutant strains of yeast. Temperature effects on the enzymes isolated from the ergosterol producing wild-type and nystatin resistant mutants (major sterol Δ⁸(9), 22 ergostadiene-3-β-ol) were compared. Transition temperatures were lower in both mutant strains compared to wild-type. Lipid analysis shows a relationship between sterol content and the temperature dependent transition phases.     

Regulation of early enzymes of ergosterol biosynthesis in Saccharomyces cerevisiae.

In order to determine the regulation mechanisms of ergosterol biosynthesis in yeast, we developed growth conditions leading to high or limiting ergosterol levels in wild type and sterol-auxotrophic mutant strains. An excess of sterol is obtained in anaerobic sterol-supplemented cultures of mutant and wild type strains. A low sterol level is obtained in aerobic growth conditions in mutant strains cultured with optimal sterol supplementation and in wild type strain deprived of pantothenic acid, as well as in anaerobic cultures without sterol supplementation. Measurements of the specific activities of acetoacetyl-CoA thiolase, HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) synthase and HMG-CoA reductase (the first three enzymes of the pathway), show that in cells deprived of ergosterol, acetoacetyl-CoA thiolase and HMG-CoA synthase are generally increased. In an excess of ergosterol, in anaerobiosis, the same enzymes are strongly decreased. A 5-10-fold decrease is observed for acetoacetyl-CoA thiolase and HMG-CoA synthase. In contrast, HMG-CoA reductase is only slightly affected by these conditions. These results show that ergosterol could regulate its own synthesis, at least partially, by repression of the first two enzymes of the pathway. Our results also show that exogenous sterols, even if strongly incorporated by auxotrophic mutant cells, cannot suppress enzyme activities in aerobic growth conditions. Measurement of specific enzyme activities in mutant cells also revealed that farnesyl pyrophosphate thwarts the enhancement of the activities of the two first enzymes.     

8(9),22 -Ergostadiene-3 -ol, an ergosterol precursor accumulated in wild-type and mutants of yeast

Whereas wild-type strains of Saccharomyces cerevisiae can synthesize up to 7% dry weight of ergosterol, a polyene-resistant mutant has been obtained which produces no ergosterol. Instead, a C-28 methyl sterol is produced, and it has been identified as Delta(8(9),22)-ergostadiene-3beta-ol. This sterol is converted to ergosterol by wild-type yeasts and is observed transiently in cells during aerobic adaption of anaerobically grown wild-type yeasts. The new sterol is proposed as an intermediate in ergosterol biosynthesis.     

Mutations in erg4 affect the sensitivity of Saccharomyces cerevisiae to medium-chain fatty acids

We have found that the medium-chain fatty acids (MCFAs) undecanoic acid (11:0), 10-undecenoic acid (11:1 Delta 10), and lauric acid (12:0) can affect the growth of Saccharomyces cerevisiae in a dose-dependent manner. The principal effect was a longer lag phase in MCFA-containing medium, although higher concentrations of 11:1 Delta 10 inhibited growth. Their relative order of inhibitory action was 11:1 Delta 10>11:0>12:0. Cellular content with MCFA supplementation was dependent on the concentration and the particular species of fatty acid, with 12:0 showing the highest relative accumulation and 11:1 Delta 10 the lowest at all concentrations. We have isolated and characterized a mutant that is hypersensitive to MCFA supplementation and is unable to grow at the normally permissive condition of 1 mM 11:1 Delta 10. However, it does not appear to accumulate higher relative levels of the fed MCFA compared to wild-type cells. Complementation of the mutant revealed that the ERG4 gene, encoding the enzyme that catalyzes the last step in ergosterol biosynthesis, had been mutated. The fatty acid composition of the erg4 Delta mutant differs only slightly from wild-type cells, mainly involving an increase in the relative amount of 12:0. These results indicate that yeast require ergosterol for optimal growth on certain MCFAs. We discuss the role ergosterol may have in cells responding to exogenous MCFAs and in supporting optimal cell growth.     

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.     

Ergosterol biosynthesis pathway in Aspergillus fumigatus

The sterol composition of Aspergillus fumigatus for the biosynthesis of ergosterol is of interest since this pathway is the target for many antifungal drugs in clinical use. The sterol composition of this fungal species was analyzed by gas chromatography-mass spectrometry in different strains (susceptible and resistant to azole drugs). Also, sterols were analyzed in several A. fumigatus mutant strains deficient in enzymatic steps of the ergosterol biosynthesis pathway such as 14-alpha sterol demethylases (Cyp51A and Cyp51B) and C-5 sterol desaturases (Erg3A, Erg3B and Erg3C). All sterols identified from azole-resistant A. fumigatus strains were qualitatively and quantitatively similar to the susceptible strain (CM-237). However, sterol composition of mutants strains were different depending on the lacking enzyme. The analysis of the sterol composition in these mutant strains led to a better understanding of the ergosterol biosynthesis pathway in this important fungus.     

Metabolism of delta24-sterols by yeast mutants blocked in removal of the C-14 methyl group.

We have investigated the metabolism of exogenously provided delta24-sterols by whole cell cultures of a polyene-resistant mutant (D10) of Candida albicans blocked at removal of the C-14 methyl group. Comparison of the relative efficiencies of transmethylation at C-24 of selected sterol substrates revealed the following substrate preferences of the Candida delta24-sterol methyltransferase (EC 2.1.1.41): zymosterol greater than 4alpha-methylzymosterol greater than 14alpha-methylzymosterol. Exogenous 4,4-dimethylzymosterol was not transmethylated by mutant D10. Incorporation of the 14C-labelled methyl group of S-adenosyl-L-[methyl-14C]methionine into the sterols of a D10 culture preloaded with zymosterol indicated that zymosterol was a better (40 X) substrate than endogenous lanosterolmfeeding zymosterol to D10 and a polyene-resistant strain of Saccharomyces cerevisiae (Nys-P100) that was also blocked at removal of the C-14 methyl group gave 24-methyl sterols possessing delta22 and ring B unsaturation. Mutant D10 was able to produce ergosterol from zymosterol whereas Nys-P100 produced ergosta-7,22-dienol. When grown in the presence of 3 micrometer 25-aza-24,25-dihydrozymosterol, a known inhibitor of the delta24-sterol methyltransferase, Nys-P100 accumulated 14alpha-methylzymosterol, a minor metabolite in this mutant under normal growth conditions and hitherto unidentified as a yeast sterol.     

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。     

Involvement of heme biosynthesis in control of sterol uptake by Saccharomyces cerevisiae.

Wild-type Saccharomyces cerevisiae do not accumulate exogenous sterols under aerobic conditions, and a mutant allele conferring sterol auxotrophy (erg7) could be isolated only in strains with a heme deficiency. delta-Aminolevulinic acid (ALA) fed to a hem1 (ALA synthetase-) erg7 (2,3-oxidosqualene cyclase-) sterol-auxotrophic strain of S. cerevisiae inhibited sterol uptake, and growth was negatively affected when intracellular sterol was depleted. The inhibition of sterol uptake (and growth of sterol auxotrophs) by ALA was dependent on the ability to synthesize heme from ALA. A procedure was developed which allowed selection of strains which would take up exogenous sterols but had no apparent defect in heme or ergosterol biosynthesis. One of these sterol uptake control mutants possessed an allele which allowed phenotypic expression of sterol auxotrophy in a heme-competent background.     

Regulation by heme of sterol uptake in Saccharomyces cerevisiae

The leaky heme mutants G204, G216, and G214 are shown to accumulate exogenous sterols. Unlike hem mutants which have complete blocks in the heme pathway, these strains do not require ergosterol, methionine, or unsaturated fatty acids for growth. The addition of aminolevulinic acid to the growth medium inhibited sterol uptake in G204 96% but had only a slight effect on sterol uptake by strains G214 and G216. Sterol uptake in all three strains was inhibited 83-94% when cells were grown in the presence of hematin. Sterol analysis of these strains grown in the presence and absence of either aminolevulinic acid or hematin revealed that saturation of the cell membrane with ergosterol was not responsible for the dramatic decrease in sterol uptake. These results suggest that sterol uptake by yeast cells is controlled by heme, and explain the non-viability of yeast strains that are heme competent and auxotrophic for sterols.     

Cholesterol import fails to prevent catalyst-based inhibition of ergosterol synthesis and cell proliferation of Trypanosoma brucei

Trypanosoma brucei (TB) cultured in rat blood, bovine serum, or lipid-depleted serum generated distinct differences in cholesterol availability. Whereas cell proliferation of the parasite was relatively unaffected by cholesterol availability, the ratios of cellular ergostenols to cholesterol varied from close to unity to 3 orders of magnitude different with cholesterol as the major sterol (>99%) of bloodstream form cells. In the procyclic form cultured with lipid-depleted serum, 15 sterols at 52 fg/cell were identified by GC-MS. The structures of these sterols reveal a nonconventional ergosterol pathway consistent with the novel product diversity catalyzed by the recently cloned sterol methyltransferase (SMT). A potent transition state analog of the TB SMT C24 alkylation reaction, 25-azalanosterol (25-AL; inhibition constant Ki = 39 nM), was found to inhibit the growth of the procyclic and bloodstream forms at an IC(50) of approximately 1 microM. This previously unrecognized catalyst-specific inhibition of cell growth was unmasked further using the 25-AL-treated procyclic form, which, compared with control cultures, caused a change in cellular sterol content from ergostenols to cholesterol. However, growth of the bloodstream form disrupted by 25-AL was not rescued by cholesterol absorption from the host, suggesting an essential role for ergosterol (24-methyl sterol) in cell proliferation and that the SMT can be a new enzyme target for drug design.     

Azasterol inhibitors in yeast. Inhibition of the 24-methylene sterol delta24(28)-reductase and delta24-sterol methyltransferase of Saccharomyces cerevisiae by 23-azacholesterol.

The effects of 23-azacholesterol on sterol biosynthesis and growth of Saccharomyces cervisiae were examined. In the presence of 0.2, 0.5, and 1 micron 23-azacholesterol, aerobically-growing yeast produced a nearly constant amount of ergosta-5,7,22,24(28)-tetraenol (approx. 36% of total sterol) and slowly accumulated zymosterol with a concommitant decline in ergosterol synthesis. Growth and total sterol content of yeast cultures treated with 0.2-1 micron 23-azacholesterol were similar to that of the control culture. Yeast cultures treated with 5 and 10 micron 23-azacholesterol produced mostly zymosterol (58-61% of total sterol), while ergosta-5,7,22,24(28)-tetraenol production declined to less than 10% of total sterol. The observed changes in the distribution of sterols in treated cultures are consistent with inhibition of 24-methylene sterol 24(28)-sterol reductase (total inhibition at 1 micron 23-azacholesterol) and of 24-sterol methyltransferase (71% inhibition at 10 micron 23-azacholesterol). Yeast cultures treated with 10 micron 23-azacholesterol were found to contain 4,4-dimethylcholesta-8,14,24-trienol and 4alpha-methylcholesta-8,14,24-trienol, which were isolated and characterized for the first time.     

Cryptococcus neoformans mitochondrial superoxide dismutase: an essential link between antioxidant function and high-temperature growth

Manganese superoxide dismutase is an essential component of the mitochondrial antioxidant defense system of most eukaryotes. In the present study, we used a reverse-genetics approach to assess the contribution of the Cryptococcus neoformans manganese superoxide dismutase (Sod2) for antioxidant defense. Strains with mutations in the SOD2 gene exhibited increased susceptibility to oxidative stress as well as poor growth at elevated temperatures compared to isogenic wild-type strains. The sod2Delta mutants were also avirulent in a murine model of inhaled cryptococcosis. Reconstitution of a sod2Delta mutant restored Sod2 activity, eliminated the oxidative stress and temperature-sensitive (ts) phenotypes, and complemented the virulence phenotype. Characterization of the ts phenotype revealed a dependency between Sod2 antioxidant activity and the ability of C. neoformans cells to adapt to growth at elevated temperatures. The ts phenotype could be suppressed by the addition of either ascorbic acid (10 mM) or Mn salen (200 muM) at 30 degrees C, but not at 37 degrees C. Furthermore, sod2Delta mutant cells that were incubated for 24 h at 37 degrees C under anaerobic, but not aerobic, conditions were viable when shifted to the permissive conditions of 25 degrees C in the presence of air. These data suggest that the C. neoformans Sod2 is a major component of the antioxidant defense system in this human fungal pathogen and that adaptation to growth at elevated temperatures is also dependent on Sod2 activity.     

The effects of temperature acclimation on membrane sterols and phospholipids of Neurospora crassa

Experiments were conducted to examine the effects of temperature acclimation on sterol and phospholipid biosynthesis in Neurospora crassa. Cultures grown at high (37 degrees C) and low (15 degrees C) temperatures show significant differences in free and total sterol content, sterol/phospholipid ratios and distribution of major phospholipid species in total lipids and two functionally distinct membrane fractions. The ratio of free sterols to phospholipids in total cellular lipids from 15 degrees C cultures was found to be about one-half that found at 37 degrees C, whereas sterol/phospholipid ratios of mitochondrial and microsomal membranes were found to be higher at the low growth temperature. Total sterol and phospholipid biosynthetic rates showed parallel reductions in cultures acclimating to a shift from 37 to 15 degrees C growth conditions. Distribution of [14C]acetate label into free sterols was significantly lower under these conditions, however; indicating an increase in the conversion rate of sterols to sterol esters at the lower temperature. Mitochondrial and microsomal membrane fractions showed distinct phospholipid distributions which also differed from total lipid distributions at the two growth temperatures. In each case there was a consistent decrease in phosphatidylcholine and a corresponding increase in phosphatidylethanolamine as growth temperatures were lowered.     

Specific sterols required for the internalization step of endocytosis in yeast

Sterols are major components of the plasma membrane, but their functions in this membrane are not well understood. We isolated a mutant defective in the internalization step of endocytosis in a gene (ERG2) encoding a C-8 sterol isomerase that acts in the late part of the ergosterol biosynthetic pathway. In the absence of Erg2p, yeast cells accumulate sterols structurally different from ergosterol, which is the major sterol in wild-type yeast. To investigate the structural requirements of ergosterol for endocytosis in more detail, several erg mutants (erg2Delta, erg6Delta, and erg2Deltaerg6Delta) were made. Analysis of fluid phase and receptor-mediated endocytosis indicates that changes in the sterol composition lead to a defect in the internalization step. Vesicle formation and fusion along the secretory pathway were not strongly affected in the ergDelta mutants. The severity of the endocytic defect correlates with changes in sterol structure and with the abundance of specific sterols in the ergDelta mutants. Desaturation of the B ring of the sterol molecules is important for the internalization step. A single desaturation at C-8,9 was not sufficient to support internalization at 37 degrees C whereas two double bonds, either at C-5,6 and C-7,8 or at C-5,6 and C-8,9, allowed internalization.     

Effect of clomiphene on fatty acids, sterols and membrane fluidity in clavine producing Claviceps purpurea strains

Clomiphene depressed the growth and enhanced clavine production of Claviceps purpurea strains 129,35 and 59. Mycelial content of 18:2 and 16:0 fatty acids decreased, whereas that of 18:1 and 18:0 acids increased. In the mutant strain 59 clomiphene, triadimefon and ergosterol stimulated the impaired function of chanoclavine cyclase. Their effect was counteracted by plant oil. Clomiphene decreased the content of total lipids (44%), triglycerides (32%), sterols (22%) and sterol/phospholipid molar ratio. The PC/PE ratio was 9X increased. Clomiphene and triadimefon enhanced membrane fluidity of protoplasts, ergosterol and oil reverted their effect.     

Sterols in erg mutants of Phycomyces: metabolic pathways and physiological effects

Phycomyces is a fungal producer of beta-carotene and other beneficial metabolites. Several erg mutants of Phycomyces, originally selected to study the effects of membrane alteration on physiological responses, have now been used to gain information about sterol biosynthesis in filamentous fungi. One mutant, H23, and its progeny were found to be blocked at episterol C-5 dehydrogenase and did not produce ergosterol or any other sterol with a conjugated Delta(5,7) diene system. This mutant showed abnormal phototropism, which was correlated with the altered sterol composition. Another mutant, H25, seems to be a regulatory mutant. All analyzed mutants synthesized ergosta-7,22,24(28)-trien-3beta-ol, demonstrating for the first time that the sterol C-22 dehydrogenase of Phycomyces is capable of recognizing sterols with a 24(28) unsaturated side chain. New evidence regarding the biogenesis of neoergosterol and phycomysterols, the potential sparking function of cholesterol, as well as the regulation of sterol biosynthesis in this fungus is also reported. Given these results, a pathway for sterol biosynthesis in Phycomyces is proposed.     

Yeast mutants deficient in heme biosynthesis and a heme mutant additionally blocked in cyclization of 2,3-oxidosqualene.

Mutants of Saccharomyces cerevisiae were isolated which were blocked in heme biosynthesis and required heme for growth on a nonfermentable carbon source. They were rho+, and grew fermentatively on ergosterol or cholesterol and Tween 80, as a source of oleic acid. Cells grown on ergosterol and Tween 80 lacked cytochromes and catalase which were restored by growth on heme. The mutants comprised five nonoverlapping complementation groups. Tetrad analysis showed that the pleiotropic properties of each of the mutants resulted from a single mutation in one of five unlinked loci (hem1 to hem5) affecting heme biosynthesis. Biochemical studies confirmed that each mutation resulted in loss of a single enzyme activity. hem1 mutants grew on delta-aminolevulinate and lacked delta-aminolevulinate synthase activity, hem2 mutants lacked delta-aminolevulinate dehydratase, and hem3 mutants uroporphyrin I synthase. Mutants in hem1, hem2, and hem3 had an additional requirement for methionine on synthetic medium supplemented with either heme or ergosterol and Tween 80, owing to a lack of sulfite reductase which contains siroheme, a modified uroporphyrin III. Since hem4 and hem5 mutants have sulfite reductase activity under all growth conditions, they are blocked after uroporphyrin III. Cell extracts of a hem4 mutant incubated with delta-aminolevulinate accumulated coproporphyrin III suggesting a block in coproporphyrinogenase, the enzyme which converts coproporphyrinogen III to protoporphyrinogen. Cells and extracts of a hem5 mutant accumulated protoporphyrin IX. Since it was the only mutant that grew on heme but not on protoporphyrin IX, a block in ferrochelatase was suggested for this strain. Mutant strains grown on heme had the sterol composition of wild type cells, whereas without heme only squalene, small amounts of lanosterol, and added sterol was observed. A heme product therefore participates in the transformation of lanosterol to ergosterol. A hem3 mutant was isolated which was also blocked between 2,3-oxidosqualene and lanosterol (erg12). When grown on lanosterol or ergosterol (with Tween 80) it accumulated a compound which was identified as 2,3-oxidosqualene by comparison with the synthetic compound in thin layer and gas-liquid chromatography, and by proton magnetic resonance and mass spectroscopy. Supplementation with heme did not remove the requirement for sterol, but it enabled the mutant to convert lanosterol to ergosterol.     

Fine measurement of ergosterol requirements for growth of Saccharomyces cerevisiae during alcoholic fermentation

Yeasts can incorporate a wide variety of exogenous sterols under strict anaerobiosis. Yeasts normally require oxygen for growth when exogenous sterols are limiting, as this favours the synthesis of lipids (sterols and unsaturated fatty acids). Although much is known about the oxygen requirements of yeasts during anaerobic growth, little is known about their exact sterol requirements in such conditions. We developed a method to determine the amount of ergosterol required for the growth of several yeast strains. We found that pre-cultured yeast strains all contained similar amounts of stored sterols, but exhibited different ergosterol assimilation efficiencies in enological conditions [as measured by the ergosterol concentration required to sustain half the number of generations attributed to ergosterol assimilation (P₅₀)]. P₅₀ was correlated with the intensity of sterol synthesis. Active dry yeasts (ADYs) contained less stored sterols than their pre-cultured counterparts and displayed very different ergosterol assimilation efficiencies. We showed that five different batches of the same industrial Saccharomyces cerevisiae ADY exhibited significantly different ergosterol requirements for growth. These differences were mainly attributed to differences in initial sterol reserves. The method described here can therefore be used to quantify indirectly the sterol synthesis abilities of yeast strains and to estimate the size of sterol reserves.     

Inhibitors of ergosterol biosynthesis and growth of the trypanosomatid protozoan Crithidia fasciculata

Six nitrogen-, sulfur- and cyclopropane-containing derivatives of cholestanol were examined as inhibitors of growth and sterol biosynthesis in the trypanosomatid protozoan Crithidia fasciculata. The concentrations of inhibitors in the culture medium required for 50% inhibition of growth were 0.32 microM for 24-thia-5 alpha,20 xi-cholestan-3 beta-ol (2), 0.009 microM for 24-methyl-24-aza-5 alpha,20 xi-cholestan-3 beta-ol (3), 0.95 microM for (20,21),(24,-25)-bis-(methylene)-5 alpha,20 xi-cholestan-3 beta-ol (4), 0.13 microM for 22-aza-5 alpha,20 xi-cholestan-3 beta-ol (5), and 0.3 microM for 23-azacholestan-3-ol (7). 23-Thia-5 alpha-cholestan-3 beta-ol (6) had no effect on protozoan growth at concentrations as high as 20 microM. Ergosterol was the major sterol observed in untreated C. fasciculata, but significant amounts of ergost-7-en-3 beta-ol, ergosta-7,24(28)-dien-3 beta-ol, ergosta-5,7,22,24(28)-tetraen-e beta-ol, cholesta-8,24-dien-3 beta-ol, and, in an unusual finding, 14 alpha-methyl-cholesta-8,24-dien-3 beta-ol were also present. When C. fasciculata was cultured in the presence of compounds 2 and 3, ergosterol synthesis was suppressed, and the principal sterol observed was cholesta-5,7,24-trien-3 beta-ol, a sterol which is not observed in untreated cultures. The presence of this trienol strongly suggests that 2 and 3 specifically inhibit the S-adenosylmethionine:sterol C-24 methyltransferase but do not interfere with the normal enzymatic processing of the sterol nucleus. When C. fasciculata was cultured in the presence of compounds 5 and 7, the levels of ergosterol and ergost-7-en-3 beta-ol were suppressed, but the amounts of the presumed immediate precursors of these sterols, ergosta-5,7,22,24(28)-tetraen-3 beta-ol and ergosta-7,24-(28)-dien-3 beta-ol, respectively, were correspondingly increased. These findings suggest that 5 and 7 specifically inhibit the reduction of the delta 24(28) side chain double bond. When C. fasciculata was cultured in the presence of compound 4, ergosterol synthesis was suppressed, but the sterol distribution in these cells was complex and not easily interpreted. Compound 6 had no significant effect on sterol synthesis in C. fasciculata.