Is Pneumocystis a Plant?

Pneumocystis, an AIDS-associated opportunistic pathogen of the lung has some unusual features. This article focuses on work done by my group to understand the organism's distinct sterols. Although Pneumocystis is closely related to fungi, it lacks the major fungal sterol, ergosterol. Several delta(7) 24-alkysterols synthesized by P. carinii are the same as those reported in some basidiomycete rust fungi. The 24-alkylsterols are synthesized by the action of S-adenosyl-L-methionine:C-24 sterol methyl transferase (SAM:SMT). Fungal SAM:SMT enzymes normally transfer only one methyl group to the C-24 position of the sterol side chain and the cells accumulate C28 24-alkylsterols. In contrast, the P. carinii SAM:SMT and those of some plants catalyze one or two methyl transfer reactions producing both C28 and C29 24-alkylsterols. However, unlike most fungi, plants, and the kinetoplastid flagellates Leishmania and Trypanosoma cruzi, P. carinii does not appear to form double bonds at C-5 of the sterol nucleus and C-22 of the sterol side chain. Furthermore, the P. carinii SAM:SMT substrate preference for C30 lanosterol differs from that of homologous enzymes in any other organisms studied. C31 24-Methylenelanosterol and C32 pneumocysterol, products of SAM:SMT activity on lanosterol, can accumulate in high amounts in some, but not all, human-derived Pneumocystis jiroveci populations.     

Structural requirements for transformation of substrates by the S-adenosyl-L-methionine:delta 24(25)-sterol methyltransferase. Inhibition by analogs of the transition state coordinate.

Microsomes from sunflower seedlings were used to investigate the transition state coordinate for the C-24 methylation reaction that mediates phytosterol biosynthesis. They were then used to study structurally related cationic and uncharged compounds of the natural sterol substrate, which were designed to interfere with the reaction progress. The hypothetical reaction course is described to proceed through an Sn2 formation of an activated complex involving the initial production of a covalent structure with a dative bond (methyl from AdoMet attacks si-face of the 24,25-double bond of the sterol) and the secondary production of a series of high energy intermediates, the stabilization of which determines the final C-24 methylated product. Derivatives of lanosterol and cholesterol with a methyl, hydrogen, oxygen, or bromine atom introduced into the side chain and/or at C-3 in place of the natural nucleophile were studied as inhibitors that interfere with the formation of the hypothetical tertiary isopropylcarbinyl cation intermediate in the conversion of cycloartenal to 24(28)-methylene cycloartanol. The data indicate the most potent inhibitor is a sterol with an aziridine group attached to C-24(25), which mimics the bridged C-24(25) carbenium ion generated in the transition state, and the methyltransferase possesses two strategic sites: one that recognizes the proximal end of the sterol acting as a proton donor and the other that recognizes the distal end that acts as a proton acceptor. The best fit (binding/catalysis) involves a flat sterol (including substrate and inhibitor) with intact unsubstituted nucleophilic centers at C-3 and C-24 and a freely rotating side chain that can assume the pseudocyclic conformation.     

Sterol composition of Pneumocystis jirovecii with blocked 14alpha-demethylase activity

Several drugs that interact with membrane sterols or inhibit their syntheses are effective in clearing a number of fungal infections. The AIDS-associated lung infection caused by Pneumocystis jirovecii is not cleared by many of these therapies. Pneumocystis normally synthesizes distinct C28 and C29 24-alkylsterols, but ergosterol, the major fungal sterol, is not among them. Two distinct sterol compositional phenotypes were previously observed in P. jirovecii. One was characterized by delta7 C28 and C29 24-alkylsterols with only low proportions of higher molecular mass components. In contrast, the other type was dominated by high C31 and C32 24-alkylsterols, especially pneumocysterol. In the present study, 28 molecular species were elucidated by nuclear magnetic resonance analysis of a human lung specimen containing P. jirovecii representing the latter sterol profile phenotype. Fifteen of the 28 had the methyl group at C-14 of the sterol nucleus and these represented 96% of the total sterol mass in the specimen (excluding cholesterol). These results strongly suggest that sterol 14alpha-demethylase was blocked in these organisms. Twenty-four of the 28 were 24-alkylsterols, indicating that methylation of the C-24 position of the sterol side chain by S-adenosyl-L-methionine:sterol C-24 methyl transferase was fully functional.     

76 The Pneumocystis sam:smt, an atypical fungal enzyme protein

Pneumocystis , an opportunistic fungal protist, causes a type of pneumonia in immunocompromised individuals such as AIDS patients. Rat-derived P. carinii and human-derived P. jiroveci contain a large number of sterols with C-24 alkyl groups. S-Adenosyl-L-methionine:sterol C-24 methyl transferase (SAM:SMT) is the enzyme that transfers methyl groups from SAM to the C-24 position of the sterol side chain. An alkyl group at the C-24 sterol side chain position appears to be essential for the organism to proliferate. Thus SAM:SMT, which is absent in mammals, is an attractive target for chemotherapeutic attack against the pathogen. The P. carinii erg6 gene that codes for SAM:SMT has been sequenced, cloned, and the protein expressed in E. coli . Since bacteria do not synthesize sterols, and do not have SAM:SMT, the P. carinii erg6 gene product expressed in E. coli would only transmethylate exogenously provided sterol substrates. The P. carinii recombinant SAM:SMT is unique because lanosterol, a central intermediate in sterol biosynthesis, is its preferred substrate for enzyme activity. Most SAM:SMT from other organisms do not bind lanosterol and prefer other sterol substrates produced from lanosterol. Furthermore, it appears that this unusual P. carinii SAM:SMT can also methylate cholesterol, which is readily scavenged from the lungs of its rat host. The recombinant enzyme protein is being purified by affinity chromatography techniques, which will be used to obtain definitive structural analyses of the sterol compounds formed by the enzyme reaction using different sterols substrates and allow detailed structural analysis of this unusual SAM:SMT enzyme protein.     

The functional importance of structural features of ergosterol in yeast.

As an approach to the study of the relationship between the structure of sterols and their capacity to function in the lipid leaflet of membranes, various sterols were examined for their ability to support the growth of anaerobic Saccharomyces cerevisiae. A marked dependence on precise structural features was observed in growth-response and morphology. Of the chemical groups which distinguish ergosterol, the main sterol of S. cerevisiae, the hydroxyl group at C-3 was obligatory, and the other groups were found to be of the following relative importance: 24beta-methyl-delta22-grouping greater than 24beta-methyl group greater than delta5,7-diene system = delta5-bond approximately or equal to no double bond. Methyl groups at C-4 and C-14 were inconsistent with activity. Consequently, the data strongly suggest that the normal biosynthetic processes removal of methyl groups from the nucleus and introduction of one in the side chain are of functional significance. A double bond between C-17 and C-20 joining the steroidal side chain to the nucleus had no deleterious effect on the growth process but only if C-22 was trans-oriented to C-13. In the cis-case no growth at all proceeded. This means the natural sterol probably acts functionally in the form of its preferred conformer in which C-22 is to the right ("right-handed") in the usual view. Since the placing of a substituent (OH or CH3) in the molecule at C-20 in such a way that it appears on the front side in the right-handed conformer completely destroyed activity, the sterol apparently presents its front face to protein or phospholipid when complexing occurs.     

Comprehensive and definitive structural identities of Pneumocystis carinii sterols

Pneumocystis causes a type of pneumonia in immunodeficient mammals, such as AIDS patients. Mammals cannot alkylate the C-24 position of the sterol side chain, nor can they desaturate C-22. Thus, the reactions leading to these sterol modifications are particularly attractive targets for the development of drugs against fungal and protozoan pathogens that make them. In the present study, the definitive structures of 43 sterol molecular species in rat-derived Pneumocystis carinii were elucidated by nuclear magnetic resonance spectroscopy. Ergosterol, Delta(5,7) sterols, trienes, and tetraenes were not among them. Most (32 of the 43) were 24-alkylsterols, products of S-adenosyl-L-methionine:C-24 sterol methyl transferase (SAM:SMT) enzyme activity. Their abundance is consistent with the suggestion that SAM:SMT is highly active in this organism and that the enzyme is an excellent anti-Pneumocystis drug target. In contrast, the comprehensive analysis strongly suggest that P. carinii does not form Delta(22) sterols, thus C-22 desaturation does not appear to be a drug target in this pathogen. The lanosterol derivatives, 24-methylenelanost-8-en-3 beta-ol and (Z)-24-ethylidenelanost-8-en-3 beta-ol (pneumocysterol), previously identified in human-derived Pneumocystis jiroveci, were also detected among the sterols of the rat-derived P. carinii organisms.     

Steroids in Porifera. II. Steroid derivatives from two sponges of the family Halichondriidae. Sokotrasterol sulfate, a marine steroid with a new pattern of side chain alkylation

A trisulfated derivative of 24,25,26,26-tetramethyl-5 alpha-cholest-23E-ene-2 alpha, 3 beta, 6 alpha-triol (sokotrasterol sulfate) has been isolated from the sponge Halichondriidae gen. sp., collected near Sokotra Island (Arabian Sea), and its structure has been elucidated. The side chain of the new steroid involves a "normal" alkylation at C-24 and the unprecedented addition of two extra methyl groups at C-26 and one extra methyl group at C-25. A free sterol fraction contained only 24-isopropyl-5-cholesten-3 beta-ol and 24-isopropyl-5, 22E-cholestadien-3 beta-ol. 24-Isopropyl-5, 22E-cholestadien-3 beta-ol as sole monohydroxy sterol and halistanol sulfate as major polyhydroxylated steroid derivative have been detected in Halichondria sp., a Madagascar sponge.     

Biochemical research elucidating metabolic pathways in Pneumocystis

Advances in sequencing the Pneumocystis carinii genome have helped identify potential metabolic pathways operative in the organism. Also, data from characterizing the biochemical and physiological nature of these organisms now allow elucidation of metabolic pathways as well as pose new challenges and questions that require additional experiments. These experiments are being performed despite the difficulty in doing experiments directly on this pathogen that has yet to be subcultured indefinitely and produce mass numbers of cells in vitro. This article reviews biochemical approaches that have provided insights into several Pneumocystis metabolic pathways. It focuses on 1) S-adenosyl-L-methionine (AdoMet; SAM), which is a ubiquitous participant in numerous cellular reactions; 2) sterols: focusing on oxidosqualene cyclase that forms lanosterol in P carinii; SAM:sterol C-24 methyltransferase that adds methyl groups at the C-24 position of the sterol side chain; and sterol 14alpha-demethylase that removes a methyl group at the C-14 position of the sterol nucleus; and 3) synthesis of ubiquinone homologs, which play a pivotal role in mitochondrial inner membrane and other cellular membrane electron transport.     

Minor sterols of marine invertebrates 37. Isolation of novel coprostanols and 4α-methyl sterols from the tunicate Ascidia nigra

The complex sterol mixture isolated from $\text{A, nigra}$ was found to contain a low level of Δ⁴-3-keto steroids, 5β-stanols and 4α-methyl sterols in addition to regular (4-demethyl) sterols. The following new marine sterols were isolated and identified using MS and 360 MHz NMR: 5β-cholest-22E-en-3β-ol, 24S-methyl-5β-cholest-22E-en-3β-ol, 24-methylene-5β-cholestan-3β-ol, both epimers at C-24 of 4α-methyl-24-ethyl-5α-cholest-22E-en-3β-ol, 4α, 22ξ, 23ξ-(or 24ξ-)trimethyl-5α-cholest-8(14)-en-3β-ol and (22S, 23S, 24S)-4α-24-dimethyl-22, 23-methylene-5α-cholestan-3β-ol. The latter sterol and 23-demethylgorqosterol have opposite configurations at C-22, C-23, and C-24; the Δ⁸⁽¹⁴⁾ sterol has an unprecedented side chain.     

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.     

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.     

Characterization of the structure of a 4-methyl-delta 8,24-cholestadien-3 beta-ol isolated from rat skin

A new sterol has been isolated from the skin of rats treated with triparanol. Its chromatographic behavior on silicic acid-Celite columns and in gas-liquid chromatographic systems indicated it to be a 4-methyl-Delta(8,24)-cholestadien-3beta-ol. The specific rotation, the delayed color reaction with Liebermann-Burchard reagent, and the nuclear magnetic resonance (NMR) data support the Delta(8(9))-unsaturation. Previous workers have shown that triparanol treatment results in an accumulation of Delta(24)-unsaturated sterols in animal tissues. Consonant with this observation, the infrared, NMR, and mass spectrometric data confirm the presence of a C-24(25) unsaturated side chain in this sterol.     

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.     

Phosphatidylcholine liposomes containing cholesterol analogues with side chains of various lengths

The effect of the length of the side chain of sterols on their interaction with phosphatidylcholine was studied by measuring the permeability properties of liposomes constituted with sterol analogues with side chains of various lengths. The sensitivities of liposomes constituted with these sterol analogues toward digitonin and polyene antibiotics were also examined.The effects of sterols on phase transition of phosphatidylcholine were examined by measuring their effects on permeability increase due to perturbation of phase equilibrium and by differential scanning calorimetry. An analogue with a short side chain, isopropyl (C-22), had a very similar effect to cholesterol in suppressing the permeability increase, suggesting that the full length of the side chain is not necessary for this effect.The permeability of egg yolk phosphatidylcholine at 42°C was suppressed as much by the analogue C-22 as by cholesterol. Androstene-3-β-ol, an analogue without a side chain, however, had little suppressive effect. Thus it is concluded that the condensing effect of sterol requires a side chain, but not the full length of side chain.Liposomes constituted with analogues having a side chain with more than 5 carbon atoms showed maximum reactivity with a polyene antibiotic, amphotericin B, whereas those constituted with analogues having a side chain with less than 4 carbon atoms showed weaker reactivity. These findings indicate that a side chain with more than 5 carbon atoms is essential for the maximum interaction of liposomes with amphotericin B. Unlike amphotericin B, filipin reacted almost equally well with liposomes containing C-22 and with those containing cholesterol. Thus the chain length of the side chain of sterol is less important for interaction of liposomes with filipin than for their interaction with amphotericin B.Liposomes containing analogues having a side chain with more than 6 carbon atoms showed maximum reactivity with digitonin. Thus for the maximum interaction of liposomes with digitonin, the side chain of sterol should be longer than 6 carbon atoms.     

Sterol methyltransferase 1 controls the level of cholesterol in plants

The side chain in plant sterols can have either a methyl or ethyl addition at carbon 24 that is absent in cholesterol. The ethyl addition is the product of two sequential methyl additions. Arabidopsis contains three genes-sterol methyltransferase 1 (SMT1), SMT2, and SMT3-homologous to yeast ERG6, which is known to encode an S-adenosylmethionine-dependent C-24 SMT that catalyzes a single methyl addition. The SMT1 polypeptide is the most similar of these Arabidopsis homologs to yeast Erg6p. Moreover, expression of Arabidopsis SMT1 in erg6 restores SMT activity to the yeast mutant. The smt1 plants have pleiotropic defects: poor growth and fertility, sensitivity of the root to calcium, and a loss of proper embryo morphogenesis. smt1 has an altered sterol content: it accumulates cholesterol and has less C-24 alkylated sterols content. Escherichia coli extracts, obtained from a strain expressing the Arabidopsis SMT1 protein, can perform both the methyl and ethyl additions to appropriate sterol substrates, although with different kinetics. The fact that smt1 null mutants still produce alkylated sterols and that SMT1 can catalyze both alkylation steps shows that there is considerable overlap in the substrate specificity of enzymes in sterol biosynthesis. The availability of the SMT1 gene and mutant should permit the manipulation of phytosterol composition, which will help elucidate the role of sterols in animal nutrition.     

Specificity of sterol-glucosylating enzymes from Sinapis alba and Physarum polycephalum.

1. Sinapis alba L. seedlings contain glycosyltransferase catalyzing the synthesis of sterol glucosides in the presence of UDPglucose as sugar donor. The major activity occurs in the membranous fraction sedimenting at 300--9000 x g. Successive treatment of the particulate enzyme fraction with acetone and Triton X-100 affords a soluble glucosyltransferase preparation which can be partly purified by gel filtration on Sephadex G-150. Molecular weight of the glucosyltransferase is 1.4 . 10(5). Apparent Km values for UDPglucose and sitosterol are 8.0 . 10(-5) M and 5.0 . 10(-6) M, respectively. 2. Comparison was made of the S. alba glucosyltransferase with a similar sterol-glucosylating enzyme isolated from non-photosynthesizing organism Physarum polycephalum (Myxomycetes). UDPglucose was the most efficient glucose donor in both cases but the enzyme from Ph. polycephalum can also utilize CDPglucose and TDPglucose. Glucose acceptors are, in case of both enzymes, sterols containing a beta-OH group at C-3 and a planar ring system (5 alpha-H or double bond at C-5). The number and position of double bonds in the ring system and in the side chain, as well as the presence of additional alkyl groups in the side chain at C-24 are of secondary importance. 3. The present results indicate that both enzymes can be regarded as specific UDPglucose:sterol glucosyltransferases. Certain differences in their specificity towards donors and acceptors of the glucosyl moiety suggest, however, a different structure of the active sites in both enzymes.     

Stereochemical specificity for sterols in Saccharomyces cerevisiae

When sterol biosynthesis in oxygen-deprived wild type Saccharomyces cerevisiae was prevented by the presence of 2,3-iminosqualene, an inhibitor of 2,3-oxidosqualene cyclase, an absolute requirement for a sterol with a 24 beta-methyl group was found. Neither the configuration nor the size of the alkyl group at C-24 could be altered. For instance, while 24 beta-methylcholesterol (22-dihydrobrassicasterol) permitted good growth, contrary to earlier work without the inhibitor no growth at all resulted from the presence of cholesterol or its 24 alpha-methyl-, 24 alpha-ethyl-, or 24 beta-ethyl derivatives (campesterol, sitosterol, and clionasterol, respectively). The only sterol lacking a 24 beta-methyl group which allowed growth was desmosterol (24-dehydro-cholesterol), but desmosterol was metabolized to 24 beta-methylcholesterol by C1-transfer and reduction. When cholesterol supported growth in the absence of the inhibitor, small amounts of endogenously synthesized 24 beta-methylsterols (ergosterol and 22-dihydroergosterol) were identified. This previously unrecognized absolute specificity for both chirality and bulk at C-24 suggests the involvement of protein binding in at least one of the roles which sterol plays in this single-celled eukaryote.     

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.     

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.