Sterol metabolism during germination of conidia of Aspergillus fumigatus.

The sterol content of germinating conidia of the opportunistic pathogenic fungus Aspergillus fumigatus has been correlated with germination phase and sensitivity to polyene antibiotics. The sterol and sterol ester contents of walls did not change during germination. The sterol ester content of membranes and cell sap remained constant during germination, whereas the sterol content increased during the outgrowth of germ tubes. On the basis of differential extraction studies it was concluded that the loss of resistance to polyenes that occurred in the early stages of swelling of conidia during germination was not due to a movement of sterol or sterol ester out of the wall. Radioactive-labelling experiments demonstrated that, although the amounts of conidial wall sterol and sterol ester did not change during germination, they were metabolically active. Changes in the turnover rate of wall and membrane sterol and sterol ester during germination were investigated and their relationship to a possible mechanism for the change from resistance to sensitivity to polyene antibiotics is discussed.     

Sterol distribution in intracellular organelles isolated from tobacco leaves

All membrane-containing fractions isolated from tobacco leaves contained free sterols, sterol glycosides, and sterol esters. The three sterol forms increased, on a dry weight basis, with a decrease in particle size. The supernatant fraction contained only trace amounts of sterol. The major sterols in all cellular fractions, in the order of decreasing amounts, were: stigmasterol, β-sitosterol, campesterol, and cholesterol. The 500g pellet contained the largest percentage of free sterol, while the 46,000g pellet contained the largest percentage of esterified sterol. The individual sterol composition of the free sterol and sterol glycoside fraction was very similar; however, the composition of the sterol ester fraction varied widely among intracellular fraction. The intracellular distribution pattern of cholesterol-¹⁴C added to the isolation medium provided evidence that the intracellular sterol distribution pattern is not an artifact. These results support the suggestion that sterols in plant cells may have a physiological function associated with membranes.     

Multiple functions for sterols in Saccharomyces cerevisiae

Analyses with a yeast sterol auxotroph indicated that there are at least four different levels of function for sterol which have been designated sparking, critical domain, domain and bulk. Growth of yeast sterol auxotrophs on cholestanol is precluded unless minute amounts of ergosterol are available. We have designated this phenomenon the sparking of growth, in which cholestanol satisfies an overall membrane sterol requirement and ergosterol fulfills a high specificity sparking function. The critical domain role for sterol is observed under conditions of lanosterol supplementation where low levels of ergosterol (10-times those necessary for sparking on cholestanol) are required for growth. The sterol functions designated domain and bulk are illustrated by assessing cellular free sterol levels and plasma membrane properties of a sterol auxotroph after growth on different concentrations of exogenously supplied sterol. Plasma membranes isolated from auxotrophs grown on domain or bulk levels of sterol underwent no lipid thermotropic transitions, while plasma membranes from cells grown on critical domain levels of sterol underwent a lipid thermotropic transition, when analyzed by steady-state fluorescence anisotropy.     

Characteristics of sterol uptake in Saccharomyces cerevisiae.

A Saccharomyces cerevisiae sterol auxotroph, FY3 (alpha hem1 erg7 ura), was used to probe the characteristics of sterol uptake in S. cerevisiae. The steady-state cellular concentration of free sterol at the late exponential phase of growth could be adjusted within a 10-fold range by varying the concentration of exogenously supplied sterol. When cultured on 1 microgram of sterol ml-1, the cells contained a minimal cellular free-cholesterol concentration of 0.85 nmol/mg (dry weight) and were termed sterol depleted. When cultured on 11 micrograms of sterol ml-1 or more, the cells contained a maximal cellular free-cholesterol concentration of 6.8 nmol/mg (dry weight) and were termed free sterol saturated. Cells with free-sterol concentrations below the maximal level were capable of accumulating free sterol from the medium. The capacity of the cells for cholesterol uptake was inversely proportional to the initial intracellular concentration. The uptake of sterol was shown to be a nonactive process that is independent of cellular energy sources or viability. The intracellular transport of sterol for esterification is not sensitive to anti-microtubule agents.     

Molecular dynamics simulation of the structure of dimyristoylphosphatidylcholine bilayers with cholesterol, ergosterol, and lanosterol

Five molecular dynamics computer simulations were performed on different phospholipid:sterol membrane systems in order to study the influence of sterol structure on membrane properties. Three of these simulated bilayer systems were composed of a 1:8 sterol:phospholipid ratio, each of which employed one of the sterol molecules: cholesterol, ergosterol, and lanosterol. The two other simulations were of a bilayer with a 1:1 sterol:phospholipid ratio. These simulations employed cholesterol and lanosterol, respectively, as their sterol components. The observed differences in simulations with cholesterol and lanosterol may have their implication on the form of the phospholipid/sterol phase diagram.     

Role of sterol superlattice in free radical-induced sterol oxidation in lipid membranes

We developed a new fluorescence assay for sterol oxidation and used it to study the relationship between free radical-induced sterol oxidation and membrane sterol lateral organization. This assay used dehydroergosterol (DHE) as both a membrane probe and a membrane component. Sterol oxidation was induced by a free radical generator, AAPH (2,2'-azobis(2-amidinopropane)dihydrochloride). Using this new assay, we found that, in unilamellar vesicles composed of DHE and 1-palmitoyl-2-oleoyl-l-alpha-phosphatidylcholine (POPC), the initial rate of DHE oxidation induced by AAPH changed with membrane sterol content in an alternating manner, exhibiting a local maximum at 20.3, 22.2, 25.0, 32.3, and 40.0 mol % DHE. These mole fractions correspond to the critical sterol mole fractions C(r) predicted for maximal sterol superlattice formation. In three-component bilayers composed of POPC, cholesterol, and DHE (fixed at 1 and 5 mol %), the initial rate of AAPH-induced DHE oxidation exhibited a biphasic change whenever the total sterol mole fraction, irrespective of the DHE content, was near C(r), indicating that the correlation between sterol oxidation and sterol superlattice formation revealed in this study is not an artifact due to the use of the fluorescent cholesterol analogue DHE. The alternating variation of AAPH-induced sterol oxidation with sterol content also appeared in multicomponent unilamellar vesicles containing bovine brain sphingomyelins (bbSPM), POPC, and DHE. The present work and our previous study on cholesterol oxidase-induced sterol oxidation [Wang et al. (2004) Biochemistry 43, 2159-2166] suggest that sterol oxidation in general, either by reactive oxygen species or by enzymes, may be regulated by the extent of sterol superlattice in the membrane and thus regulated by the membrane sterol content in a fine-tuning manner.     

Quantification of sterol lipids in plants by quadrupole time-of-flight mass spectrometry

Glycerolipids, sphingolipids, and sterol lipids constitute the major lipid classes in plants. Sterol lipids are composed of free and conjugated sterols, i.e., sterol esters, sterol glycosides, and acylated sterol glycosides. Sterol lipids play crucial roles during adaption to abiotic stresses and plant-pathogen interactions. Presently, no comprehensive method for sterol lipid quantification in plants is available. We used nanospray ionization quadrupole-time-of-flight mass spectrometry (Q-TOF MS) to resolve and identify the molecular species of all four sterol lipid classes from Arabidopsis thaliana. Free sterols were derivatized with chlorobetainyl chloride. Sterol esters, sterol glycosides, and acylated sterol glycosides were ionized as ammonium adducts. Quantification of molecular species was achieved in the positive mode after fragmentation in the presence of internal standards. The amounts of sterol lipids quantified by Q-TOF MS/MS were validated by comparison with results obtained with TLC/GC. Quantification of sterol lipids from leaves and roots of phosphate-deprived A. thaliana plants revealed changes in the amounts and molecular species composition. The Q-TOF method is far more sensitive than GC or HPLC. Therefore, Q-TOF MS/MS provides a comprehensive strategy for sterol lipid quantification that can be adapted to other tandem mass spectrometers.     

Xylanase treatment of plant cells induces glycosylation and fatty acylation of phytosterols

Treatment of tobacco suspension cells ( Nicotiana tabacum cv. KY 14) with a purified β -1,4-endoxylanase from Trichoderma viride [1 μg enzyme (ml cells)⁻¹] caused a 13-fold increase in the levels of acylated sterol glycosides and elicited the synthesis of phytoalexins. A commercial preparation of xylanase from Trichoderma viride caused an identical shift in sterols. In contrast, a commerical xylanase from Aureobasidium pullaulans had no effect on the levels of acylated sterol glycosides, but did elevate the levels of sterol esters. Treatment of the cells with Cu²⁺ or Ag⁺ also evoked a severalfold increase in the levels of acylated sterol glycosides. Analysis of the various sterol lipid classes revealed that the large xylanase-induced increase in acylated sterol glycosides occurred at the expense of sterol esters, free sterols and sterol glycosides. Further analyses revealed that the most abundant phytosterol in each of the four classes of sterol lipids was β -sitosterol. Linoleic acid was the most abundant fatty acid in the sterol esters, and palmitic and linoleic acids were the most abundant fatty acids in the acylated sterol glycosides. Glucose was the only sugar moiety in the sterol glycoside and acvlated sterol glycosides. Glucose was the only sugar moiety in the sterol glycoside and acylated sterol glycoside fractions. The results of the present study demonstrate that xylanase from Trichoderma viride induces a dramatic shift in the level of acylated sterol glycosides, indicating that endoxylanase was probably the active component in the cellulase enzyme preparations used in our previous study.     

STEROLS OF CEPHALEUROS (TRENTEPOHLIACEAE), A PARASITIC GREEN ALGA1

24-Ethylcholesterol, 24-ethylcholesta-5,7,22-trienol, dihydrolanosterol, 24-ethylcholesta-7,22-dienol, and 4-methyl-24-ethylcholesta-7,22-dienol were identified in cultured Cephaleuros in small quantities. Cholesterol made up 19% of the total sterol. The principal sterol, making up 65% of the total sterol, was 4,24-dimethylcholest-7-enol, a new algal sterol. This is the first report of this sterol as the principal sterol of any living organism.     

A comparison of the packing behavior of egg phosphatidylcholine with cholesterol and biogenically related sterols in Langmuir monolayer films

Cholesterol and selected derivatives were studied as mixed Langmuir monolayers with egg phosphatidylcholine (PC). As an extension of our earlier work, which employed binary sterol/PC mixtures, here we examined ternary mixed monolayers containing cholesterol along with an alternate sterol and PC in different molar ratios, using pressure-area isotherms. The ternary systems behaved similarly to the binary sterol/PC systems reported previously, with similar condensation noted for the sterol/PC films. To better understand how variations in sterol structure affect sterol packing in such membrane monolayers, binary mixtures containing cholestenone, cholestanol, and lanosterol with PC were also studied. Cholestanol behaved similarly to cholesterol when incorporated with PC, while cholestenone and lanosterol did not cause as much film condensation. The observed differences in molecular packing, and attributed sterol structural differences, are considered within the context of sterol/phospholipid mixtures in biological membranes.     

Mutations in yeast ARV1 alter intracellular sterol distribution and are complemented by human ARV1

Intracellular cholesterol redistribution between membranes and its subsequent esterification are critical aspects of lipid homeostasis that prevent free sterol toxicity. To identify genes that mediate sterol trafficking, we screened for yeast mutants that were inviable in the absence of sterol esterification. Mutations in the novel gene, ARV1, render cells dependent on sterol esterification for growth, nystatin-sensitive, temperature-sensitive, and anaerobically inviable. Cells lacking Arv1p display altered intracellular sterol distribution and are defective in sterol uptake, consistent with a role for Arv1p in trafficking sterol into the plasma membrane. Human ARV1, a predicted sequence ortholog of yeast ARV1, complements the defects associated with deletion of the yeast gene. The genes are predicted to encode transmembrane proteins with potential zinc-binding motifs. We propose that ARV1 is a novel mediator of eukaryotic sterol homeostasis.     

Changes in Sterol Composition during Greening of Etiolated Barley Shoots

The following sterols were identified in barley shoots: stigmasterol, β-sitosterol, campesterol, and cholesterol. The total sterol content of green and etiolated tissue was 2.84 and 3.20 milligrams per gram dry weight, respectively. The free sterols accounted for most of the difference in total sterol content. The sterol ester, sterol glycoside, and acylated sterol glycoside contents of green and etiolated barley shoots were essentially the same. Etiolated tissue had twice as much total β-sitosterol as stigmasterol, while green tissue had equal amounts of these two sterols. The campesterol and cholesterol content was the same in green and etiolated tissue. This same sterol composition pattern held true for the free, glycosidic, and acylated glycosidic sterols; however, the sterol ester fraction had a completely different composition pattern. The esterified stigmasterol content was quite low in green and etiolated tissue, and campesterol was the second largest esterfied sterol component in etiolated tissue. Etiolated barley seedlings exposed to light had a shift in the ratio of free stigmasterol to β-sitosterol in favor of stigmasterol; however, no correlation was observed between chlorophyll synthesis and shift in sterol composition.     

ATP-binding cassette (ABC) transporters mediate nonvesicular, raft-modulated sterol movement from the plasma membrane to the endoplasmic reticulum

Little is known about the mechanisms of intracellular sterol transport or how cells maintain the high sterol concentration of the plasma membrane (PM). Here we demonstrate that two inducible ATP-binding cassette (ABC) transporters (Aus1p and Pdr11p) mediate nonvesicular movement of PM sterol to the endoplasmic reticulum (ER) in Saccharomyces cerevisiae. This transport facilitates exogenous sterol uptake, which we find requires steryl ester synthesis in the ER. Surprisingly, while expression of Aus1p and Pdr11p significantly increases sterol movement from PM to ER, it does not alter intracellular sterol distribution. Thus, ER sterol is likely rapidly returned to the PM when it is not esterified in the ER. We show that the propensity of PM sterols to be moved to the ER is largely determined by their affinity for sterol sphingolipid-enriched microdomains (rafts). Our findings suggest that raft association is a primary determinant of sterol accumulation in the PM and that Aus1p and Pdr11p facilitate sterol uptake by increasing the cycling of sterol between the PM and ER.     

Deficiency in the Lipid Exporter ABCA1 Impairs Retrograde Sterol Movement and Disrupts Sterol Sensing at the Endoplasmic Reticulum

Cellular cholesterol homeostasis involves sterol sensing at the endoplasmic reticulum (ER) and sterol export from the plasma membrane (PM). Sterol sensing at the ER requires efficient sterol delivery from the PM; however, the macromolecules that facilitate retrograde sterol transport at the PM have not been identified. ATP-binding cassette transporter A1 (ABCA1) mediates cholesterol and phospholipid export to apolipoprotein A-I for the assembly of high density lipoprotein (HDL). Mutations in ABCA1 cause Tangier disease, a familial HDL deficiency. Several lines of clinical and experimental evidence suggest a second function of ABCA1 in cellular cholesterol homeostasis in addition to mediating cholesterol efflux. Here, we report the unexpected finding that ABCA1 also plays a key role in facilitating retrograde sterol transport from the PM to the ER for sterol sensing. Deficiency in ABCA1 delays sterol esterification at the ER and activates the SREBP-2 cleavage pathway. The intrinsic ATPase activity in ABCA1 is required to facilitate retrograde sterol transport. ABCA1 deficiency causes alternation of PM composition and hampers a clathrin-independent endocytic activity that is required for ER sterol sensing. Our finding identifies ABCA1 as a key macromolecule facilitating bidirectional sterol movement at the PM and shows that ABCA1 controls retrograde sterol transport by modulating a certain clathrin-independent endocytic process.     

Modulation of yeast plasma membrane composition of a yeast sterol auxotroph as a function of exogenous sterol

Plasma membranes isolated from a yeast sterol auxotroph (RD5-R) grown on 1, 5, and 15 micrograms ml-1 exogenous concentrations of sterol showed no discontinuity in plots of steady-state fluorescence anisotropy. Liposomes constructed from phospholipid and sterol extracted from RD5-R grown on different sterols indicated that exogenously supplied sterol modulated cellular phospholipids such that lipid-phase transitions were avoided. Liposomes derived from sterol and phospholipid extracted from the same culture exhibited no lipid-phase transitions. However, when phospholipid extracted from a culture grown on a specific sterol was mixed with sterol extracted from a heterologous culture grown on a different sterol to form liposomes, discontinuities were detected in the anisotropy measurements of the liposomes produced. Quantitative analyses revealed that the exogenously supplied sterol coordinately regulated specific phospholipid species, fatty acid composition, and sterol to phospholipid ratios in yeast auxotrophs.     

Multiple roles for lipids in the Hedgehog signalling pathway

The identification of endogenous sterol derivatives that modulate the Hedgehog (Hh) signalling pathway has begun to suggest testable hypotheses for the cellular biological functions of Patched, and for the lipoprotein association of Hh. Progress in the field of intracellular sterol trafficking has emphasized how tightly the distribution of intracellular sterol is controlled, and suggests that the synthesis of sterol derivatives can be influenced by specific sterol-delivery pathways. The combination of this field with Hh studies will rapidly give us a more sophisticated understanding of both the Hh signal-transduction pathway and the cell biology of sterol metabolism.     

Characterization of human sterol 27-hydroxylase. A mitochondrial cytochrome P-450 that catalyzes multiple oxidation reaction in bile acid biosynthesis.

The enzyme sterol 27-hydroxylase catalyzes the first step in the oxidation of the side chain of sterol intermediates in the bile acid synthesis pathway. Human sterol 27-hydroxylase cDNAs were isolated from a liver cDNA library by cross-hybridization with a previously cloned rabbit cDNA probe. DNA sequence analysis of hybridization-positive clones predicted a human sterol 27-hydroxylase consisting of a 33-amino-acid mitochondrial signal sequence followed by a mature protein of 498 amino acids. RNA blotting experiments demonstrated sterol 27-hydroxylase mRNAs of approximately 1.8 to 2.2 kilobases in liver and fibroblast cells. The steady state levels of the mRNA did not change when cultured cells were grown in the presence or absence of sterols. Introduction of the sterol 27-hydroxylase cDNA into Simian COS cells resulted in the expression of active enzyme capable of catalyzing multiple oxidation reactions (R-CH3----R-CH2OH----R-COOH) at carbon 27 of sterol intermediates of the bile acid synthesis pathway.     

Specific fragmentation of natural inhibitor of mitochondrial ATPase by thrombin

The ability of cholestanol (5α-cholestan-3β-ol) to support growth of two independently derived sterol auxotrophs, FY3 and GL7, has been examined. Growth on this stanol was precluded unless minute quantities of sterol were also available. Contaminating sterol in most cholestanol preparations or excess sterol in the inoculum used in growth studies could provide the required sterol in quantities capable of sustaining growth through an entire culture cycle. Evidence is presented for multiple functions of sterols in $\text{Saccharomyces}\text{cerevisiae}$.     

Origin of sterols in uredospores of Uromyces phaseoli

The sterols from healthy bean leaves are β-sitosterol, stigmasterol, campesterol and 28-isofucosterol. An additional sterol observed in bean leaves infected with Uromyces phaseoli was identified as 7,(Z)-24(28)-stigmastadien-3β-ol, which is the major sterol of the uredospores of the fungus. The fungus appears to stimulate sterol synthesis, but most of the increased sterol content of infected leaves can be attributed to the sterol of the uredospores.