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.

Evidence for facilitated transport in the absorption of sterols by Saccharomyces cerevisiae.

Saccharomyces cerevisiae is known to absorb sterols readily in the absence of air. As shown in this paper, yeast cells also will absorb sterols with and without various double bonds or an alkyl group at C-24 in the presence of air at a concentration (ca. 10% of the gas phase) which is growth-limiting due to limited sterol synthesis. However, if the growth conditions are changed to be fully aerobic, sterol is no longer absorbed to any significant extent even when the sterol in the medium (ergosterol) is the same as that present in the cells. This implies that sterol in the medium does not equilibrate passively with sterol in the plasma membrane and that some sort of facilitated transport, which can be turned on and off, is responsible for the entry of sterol when it occurs as a response to an inadequate endogenous supply of sterol. In agreement with facilitated transport mediated by protein binding, yeast cells in an auxotrophic state for sterol exhibit a high degree of stereoselectivity with respect to the orientation of the side chain around the C-17(20)-bond. For instance, E-17(20)- but not Z-17(20)-dehydrocholesterol is absorbed by cells undergoing limited growth with 10% air.

Inhibition of sterol biosynthesis by ergosterol and cholesterol in Saccharomyces cerevisiae.

When accumulation of squalene was used as a measure of the flow of carbon into the sterol pathway in whole cells of semi-anaerobic Saccharomyces cerevisiae, both ergosterol and cholesterol were found to be inhibitory. However, at equivalent concentrations in the medium ergosterol was substantially the more potent inhibitor. Marked differences found in the absorption and esterification of the two sterols failed to account for the observed difference in their capacities to act as feedback agents. Cholesterol was much more effectively absorbed as well as esterified, but, when the abilities of the two sterols to lower the squalene level were calculated on the basis of free sterol in the cells, ergosterol remained more effective by a factor of four.

Rotational isomerism about the 17(20)-bond of steroids and euphoids as shown by the crystal structures of euphol and tirucallol.

The influence of configuration at C-20 on rotation about the 17(20)-bond in steroids and euphoids was examined by x-ray crystallographic studies of the C-20 epimers euphol and tirucallol. The H atom on C-20 was in back next to C-18 in the crystal structures of both of the compounds, and C-22 was found to be cis-oriented ("left-handed") to C-13 in euphol and trans-oriented to it ("right-handed") in tirucallol. The results, which are consistent with the known left-handed crystal structure of 24(25)-dihydroeuphol and right-handed crystal structure of cholesterol and other natural sterols, lend further credence to the earlier suggestion that rotational isomerism at the 17(20)-bond can arise in C-20 epimers and that there is preference for an arrangement with the 20-H atom adjacent to C-18.

Stereochemically distinct roles for sterol in Saccharomyces cerevisiae.

Cholesterol, (E)- but not (Z)-17(20)-dehydrocholesterol, 5 alpha-cholestan-3 beta-ol, sitosterol, and certain other sterols lacking a 24 beta-methyl group will replace (spare) most but not all of the 24 beta-methylsterol which has recently been found to be absolutely necessary for growth of oxygen-deprived wild type Saccharomyces cerevisiae in the presence of 2,3-iminosqualene. The results imply the existence of two stereochemically distinct roles for sterol in this organism. One of them (perhaps regulatory) requires, whereas the other (probably playing the so-called "bulk" membranous role) does not require the presence of the 24 beta-methyl group. The latter function, for which most of the sterol is needed, can be performed by various 24-alkyl- and 24-desalkylsterols.

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.

Inhibition of hepatic cholesterol synthesis in mice by sterols with shortened and stereochemically varied side chains.

Mice were fed cholesterol or various other sterols for 26 hr, after which the amount of hepatic cholesterol synthesis was measured in a cell-free system. The following sterols were as effective as cholesterol itself in depressing the conversion of acetate into sterol: pregn-5-en-3 beta-ol, which lacks an isohexyl group on C-20; (E)-17(20)-dehydrocholesterol, in which the isohexyl group is fixed to the right; (E)-20(22)-dehydrocholesterol, in which C-23 is oriented away from the nucleus; and 20-epicholesterol. Moreover, when the isohexyl group was fixed to the left in (Z)-17(20)-dehydrocholesterol, this dietary sterol, identified in the liver, caused not only a depression in the conversion of both mevalonate and squalene into sterols. The incorporation of acetate into fatty acids was not depressed, nor did the (Z)-sterol appear to have a generalized effect on membranous enzymes, because the activity of glucose-6-phosphatase was unaffected. Thus, feedback inhibition was retained when the stereochemistry of cholesterol's side chain was drastically changed and even after the nearly complete removal of the side chain. This implies that the side chain is only minimally recognized by the mechanisms involved in feedback inhibition.

The sterol substrate specificity of acyl CoA: :cholesterol acyltransferase from rat liver.

Rat liver microsomes were incubated with various sterols suspended in Triton WR-1339, and the extent of esterification of these sterols by acyl CoA:cholesterol acyltransferase was determined. A 3 beta-hydroxyl group was required for esterification to occur. Furthermore, the rate of ester formation of campesterol was only 20% that of cholesterol, and the rates for sitosterol and stigmasterol were below detectable limits indicating that the structure of the alkyl side chain plays an important role in the interaction between substrate and enzyme. Additional evidence concerning the importance of the side chain was obtained by following the esterification of a series of linear side chain analogues of cholesterol. Maximal ester formation was obtained when the longest chain on C-20 had five carbons (the same as cholesterol) and either an increase or decrease in the number of carbons reduced the amount of ester formed. Sterols containing a 4-gem-dimethyl group were not esterified, while 4 alpha-methylcholest-7-en-3 beta-ol showed significant esterification. Lathosterol, cholestanol, and desmosterol were esterified 41%, 70%, and 62%, respectively, as well as was cholesterol. The relationship between the specificity of acyl CoA:cholesterol acyltransferase and the occurrence of sterol esters in tissues is discussed.

A comparison of the biological properties of androst-5-en-3 beta-ol, a series of (20R)-n-alkylpregn-5-en-3 beta-ols and 21-isopentylcholesterol with those of cholesterol.

The delta 5-sterol, androst-5-en-3 beta-ol, which has no side chain at C-17, did not permit molting of the insect Heliothis zea, growth of either the protozoan Tetrahymena pyriformis, or the yeast Saccharomyces cerevisiae adapted to anaerobic conditions, nor was the sterol esterified by a mammalian microsomal ACAT preparation. However, the sterol did form a liposome with egg lecithin and, when fed to mice, did inhibit hepatic cholesterol synthesis. 21-Isopentylcholesterol also formed a liposome but neither supported the growth of the yeast nor was metabolized by the protozoan. When sterols, 20(R)-n-alkylpregn-5-en-3 beta-ols, with side chains of varying lengths were added to the medium of the protozoan, maximal esterification with fatty acids occurred with the 20(R)-n-pentyl derivative, and maximal inhibition of tetrahymanol formation occurred with the n-butyl, n-pentyl, and n-hexyl derivatives. In all of the assays cholesterol showed a positive response, either permitting molting or growth, being metabolized, inhibiting sterol or tetrahymanol synthesis, or forming a liposome.

Steric effects at C-20 and C-24 on the metabolism of sterols by Tetrahymena pyriformis.

Cultures of Tetrahymena pyriformis were incubated with various sterols and the extent of dehydrogenation at C-7 and C-22 was determined. The sterols incubated were desmosterol, 22-dehydrodesmosterol, 24-methyldesmosterol, 24 alpha-methylcholesterol (campesterol), 24-methylene-cholesterol, isohalosterol (26,27-bisnorcampesterol, also known as 24,24-dimethylchol-5-en-e beta-ol, a naturally occurring C26-sterol), and 20-isohalosterol. 20-Isohalosterol was not metabolized, while products with delta 7- and delta 22-bonds were formed from isohalosterol and all of the other sterols studied. This confirms an earlier conclusion, based on results with 20-isocholesterol and cholesterol, that inversion of the configuration from 20(R) to 20(S) completely prevents metabolism both in the nucleus and the side chain. On the other hand, changes in the electronics or stereochemistry at C-24 had a direct affect only on metabolism in the side chain. The presence of a methyl group at C-24 reduced the yield of metabolites with a delta 22-bond relative to those with a delta 7-bond producing an accumulation of 7-dehydro metabolite. A double bond at position-24 counteracted this steric effect, presumably by enhancing the rate of dehydrogenation, and a delta 24(28)-bond was more effect than was a delta 24(25)-bond.

Metabolism of sterols by anaerobic Saccharomyces cerevisiae.

Anaerobically grown Saccharomyces cerevisiae retained the ability to transfer a C1-group to the C-24 position of a delta 24(25)-sterol and to reduce the delta 25(28)-bond of a 24-methylenesterol. Both desmosterol and 24-methylenecholesterol yielded 24 beta-methylcholesterol. However, when the substituent at C-24 was enlarged to a 24-ethylidene group (fucosterol), reduction of the delta 24(28)-bond did not occur. In no cases was a delta 7- or a delta 22-bond introduced. Because the delta 24(28)-bond was reduced in the absence of the delta 22-bond, the delta 22-bond is not an obligatory requirement for reduction.

Growth characteristics of Saccharomyces cerevisiae and Aspergillus nidulans when biotin is replaced by aspartic and fatty acids.

When either aerobic or anaerobic cultures of Saccharomyces cerevisiae were supplemented with aspartic and fatty acids in place of biotin, stationary phase populations were very small compared with those obtained in the presence of biotin. Similarly, these acids failed to fulfil the role of biotin-requiring strain of Aspergillus nidulans. Furthermore, a requirement for saturated fatty acid was found with anaerobically cultured S. cerevisiae. Cells were fragmented when biotin was replaced by aspartic and oleic acids alone, while cellular integrity was maintained, but with only slight growth, when biotin was replaced by oleic and palmitic acids together with aspartate. The importance of biotin in the growth of A. nidulans was particularly pronounced in the presence of glucose. In a medium containing glucose, growth ceased when biotin was replaced by aspartate and Tween 80 (a source of saturated and unsaturated fatty acids), but such replacement permitted a very small amount of growth to occur in the absence of glucose.

The effects of branching, oxygen, and chain length in the side chain of sterols on their metabolism by Tetrahymena pyriformis.

The ciliated protozoan Tetrahymena pyriformis was incubated with delta 5-sterols which has side chains varying in degree of branching, polarity, and length. Branching at neither end of the side chain was obligatory for metabolism, although removal of C-21 did have a small quantitative effect. Both 21-norcholesterol and sterols lacking a terminal gem-dimethyl group, e.g. 26 (or 27)-norcholesterol, underwent conversion to the corresponding delta 5,7,22-trienols. However, replacement of C-21 with oxygen (20 xi-hydroxy- and 20-keto-21-norcholesterol) was very deleterious. In particular, introduction of a delta 22-bond was abolished, and dehydrogenation in Ring B was strongly reduced. The overall length of the side chain which permitted maximal metabolism corresponded closely to that in cholesterol and other natural sterols. Maximal metabolism was observed only with sterols bearing side chains which had 5 or 6 carbon atoms (other than C-21) attached to C-20. The extent of metabolism fell gradually to zero as the length of the chain was increased or decreased. No metabolism at all occurred with side chains of no carbon atoms (pregn-5-en-3 beta-ol) or as many as 12 carbon atoms (20(R)-n-dodecylpregn-5-en-3 beta-ol) on C-20 other than C-21. Dehydrogenation in the side chain was more sensitive to chain length than was dehydrogenation in Ring B. The data presented here reinforce the view that the enzymes involved in sterol metabolism are actually directed toward binding with sterols. Furthermore, since the sterols become components of the ciliary membrane of this protozoan, the observed structural requirements for metabolism presumably reflect the structural requirements for membrane architecture; and the evidence presented then suggests that the natural length of the sterol side chain is governed by the sterol's membranous function.

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.

Determination of the absolute configuration at C-20 and C-24 of ergosterol in Ascomycetes and Basidiomycetes by proton magnetic resonance spectroscopy.

Samples of ergosterol isolated from Saccharomyces cerevisiae, Neurospora crassa, and Agaricus sp., and commercial ergosterol all displayed identical proton magnetic resonance (PMR) spectra at 220 MHz. From the effects produced on the doublet for C-21 by epimerization at C-20 and C-24 in sterols of known configuration, the absolute configurations at these positions in ergosterol were determined. The data demonstrate that ergosterol from both Ascomycetes and Basidiomycetes is the same and that at C-20 and C-24, the two H-atoms are on the alpha-side of the asymmetric carbon atoms and that C-22 is trans-oriented with respect to C-13 about the 17(20)-bond.

Delayed conversion of squalene to sterols during development of Pinus pinea seeds.

During germination of seeds of the gymnosperm, Pinus pinea, radioactivity from [2-14C]-mevalonate proceeded principally through the anaerobic reactions leading to squalene in the first 24 hr in both the haploid endosperm and the diploid embryo, and only with succeeding time (3-9 days) in both cases was label transferred to sterols in oxygen-requiring steps. The rates of turnover must be real and independent in the two tissues, since no consequential interchange of labelled lipids occurred between the endosperm and the embryo. Similar delayed conversion of squalene to sterols has been observed previously during germination of seeds of the angiosperm, Pisum sativum.