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L W Parks

Publications and source records attributed to L W Parks.

At least 55 records · Page 3Linked to original sources
PubMed1988

Pleiotropic mutations in Saccharomyces cerevisiae affecting sterol uptake and metabolism.

Sterol uptake control mutants (upc-) have been isolated via ethylmethanesulfonate mutagenesis from wild-type Saccharomyces cerevisiae. These mutants are heme and sterol competent but possess the ability to accumulate exogenous sterol(s) under aerobic conditions. Previous studies demonstrate sterol uptake only in a hem-, erg- background; however, the Upc- strains described here are Hem+ and do not require exogenous sterol for growth. We were unable to obtain viable hem+, erg-, upc+ recombinants; such combinations appear to be lethal. Isolates of Upc mutants demonstrated different levels of sterol uptake, and sterol analysis revealed a broad phenotypic range with regard to amounts and accumulation of ergosterol and non-ergosterol sterols. Assays of acyl CoA: ergosterol acyltransferase and sterol ester hydrolase showed no apparent difference in activity between Upc mutants and the wild type.

Cholesterol↗
PubMed1988

Purification and characterization of plasmid-like DNA from the antimycotic producing fungus, Scytalidium flavo-brunneum.

The azasterol producing strain of Scytalidium flavo-brunneum (ATCC 28804) was examined for the presence of a plasmid-like DNA. Several different plasmid preparation procedures yielded DNA which migrated as single bands of equivalent molecular weight when analyzed by gel electrophoresis. Electron microscopy and lambda exonuclease digestion data were consistent with a covalently closed circular structure. A complete restriction map for a circular 9.1-kb plasmid-like DNA was deduced from analysis of restriction enzyme digests and Southern blot hybridizations of restriction fragments. Visualization of the plasmid by electron microscopy revealed a measured contour length of 8.9 kb, using pBR322 as a standard. Southern hybridization analysis using plasmid-like DNA as the probe detected no homology to the non-azasterol producing strains of Scytalidium flavo-brunneum or mitochondrial DNA from azasterol producing strain.

Blotting, Southern↗
PubMed1987

Relationship between intracellular sterol content and sterol esterification and hydrolysis in Saccharomyces cerevisiae.

The relationship between the supply of free sterol and the synthesis of steryl esters by an auxotroph of Saccharomyces cerevisiae has been examined in order to understand the role of cellular free sterol content in the regulatory interactions of sterol esterification. Our results show that the yeast cells must maintain an essential, low level of free sterol that is critical for growth. An additional, expandable pool of free sterol is maintained by the cells, provided there is adequate available sterol. As the quantity of sterol in the expandable pool increases, there is a progressively increasing rate of sterol esterification, which is consistent with the results from in vitro assays of acyl-Co A:ergosterol acyltransferase. Some increases in acyltransferase activity were insensitive to the protein synthetic inhibitor, cycloheximide. The data support the conclusion that sterol interconversion between the free and esterified forms is directed toward maintaining the essential amount of free sterol and that the activity of the relevant sterol enzymes in this organism are modulated in response to intracellular sterol content.

Acyltransferases↗
PubMed1987

Sterol and phospholipid acyl chain alterations in Saccharomyces cerevisiae secretion mutants as a function of temperature stress.

Analyses of free sterol, steryl ester and fatty acid components from yeast secretion mutants indicated that free and esterified sterol remained relatively constant over a growth range of 24 C to 34 C. The saturated fatty acid components (16:0 and 18:0) increased while the unsaturated fatty acids (16:1 and 18:1) decreased as the growth temperature increased. In secretory mutants, fatty acid composition changes are more pronounced than in the wild-type strain. A shift toward increased saturated and decreased unsaturated fatty acid was observed when cells were subjected to a 2-hr temperature upshift to 37 C. Steady-state fluorescence anisotropy data indicated that modifications to the lipid component of yeast plasma membrane produced lipid thermotropic transitions that were 3 C to 6 C higher in yeast cells subjected to thermal stress.

Acylation↗
PubMed1987

Autoconditioning factor relieves ethanol-induced growth inhibition of Saccharomyces cerevisiae.

Viable Saccharomyces cerevisiae suspended in medium containing growth-inhibiting concentrations of ethanol produce a metabolite that relieves growth inhibition. This autoconditioning of the medium by yeasts is due to the formation of small amounts (0.01%, vol/vol) of acetaldehyde. The effect is duplicated precisely in fresh medium by the addition of acetaldehyde. Acetaldehyde does not increase the yield of or accelerate ethanol production by the organism. Ethanol-induced modifications of membrane order in the plasma membranes, as measured by steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene, were not resolved by exogenously added acetaldehyde.

Acetaldehyde↗
PubMed1987

Regulation of ergosterol biosynthesis and sterol uptake in a sterol-auxotrophic yeast.

Inhibition of sterol uptake in Saccharomyces cerevisiae sterol auxotroph FY3 (alpha hem1 erg7 ura) by delta-aminolevulinic acid (ALA) is dependent on the ability of the organism to synthesize heme from ALA. Sterol-depleted cells not exposed to ALA or strain PFY3 cells, with a double heme mutation, exposed to ALA did not exhibit inhibition of sterol uptake. Addition of ALA to sterol-depleted FY3 stimulated production of a high endogenous concentration of 2,3-oxidosqualene (25.55 micrograms mg-1 [dry weight]) at 24 h, whereas FY3 not exposed to ALA or PFY3 exposed to ALA did not accumulate 2,3-oxidosqualene. The high concentration of 2,3-oxidosqualene in FY3 with ALA decreased, and 2,3;22,23-dioxidosqualene increased to a very high level. The elevation of 2,3-oxidosqualene by ALA was correlated with a fivefold increase in the activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (EC 1.1.1.34). The enhanced activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase was prevented by cycloheximide but not chloramphenicol and was dependent on a fermentative energy source. Inhibition of sterol uptake could not be attributed to 2,3-oxidosqualene or 2,3;22,23-dioxidosqualene but was due to a nonsaturating level of ergosterol produced as a consequence of heme competency through a leaky erg7 mutation.

Aminolevulinic Acid↗
PubMed1986

An essential fungal growth factor derived from ergosterol: a new end product of sterol biosynthesis in fungi?

Concentrations of ergosterol as low as 1.2 mM are effective in satisfying the essential high specificity microrequirement for C5,6 unsaturated sterol in yeast. In a sterol auxotroph supplemented with saturated sterol (cholestanol), aqueous extracts of yeast provide a growth factor that eliminates the ergosterol microrequirement. The factor is easily differentiated from ergosterol by solubility, thermostability, and thin layer and liquid chromatography and is functional at levels equivalent to those of vitamins, hormones and pheromones.

Cholestanols↗
PubMed1986

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.

Biological Transport↗
PubMed1985

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.

Fluorescence Polarization↗
PubMed1985

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.

Cell Membrane↗
PubMed1985

Influence of sterol structure on yeast plasma membrane properties.

Fluorescence anisotropy measurements indicated that physical changes occurred in the lipids of plasma membranes of yeast sterol mutants but not in the plasma membrane of an ergosterol wild-type. Parallel experiments with model membrane liposomes verified that the physical changes in lipids observed in the sterol mutants are dependent on the sterol present and not the phospholipid composition. In addition, the physical changes in lipids observed in liposomes derived from wild-type phospholipids were eliminated by addition of ergosterol but persisted in the presence of cholesterol, cholestanol, ergostanol, or sterols from the sterol mutants. No physical changes in lipids were observed, however, in plasma membranes from a sterol auxotroph, even when the auxotroph was grown on cholesterol or cholestanol. The lack of physical changes in lipids in the sterol auxotroph may reflect the ability of the auxotroph to modify its phospholipid composition with respect to its sterol composition. These results indicate that high specificity 'sparking' sterol is not required for the regulation of overall bulk lipid properties of the plasma membrane.

Cell Membrane↗
PubMed1985

High-performance liquid chromatography of sterols: yeast sterols.

It is evident that the high-pressure liquid chromatograph is an excellent tool for studying sterol metabolism. As noted in the text, the individual effects of unsaturations and alkyl groups on reverse phase elution volumes cannot be extrapolated to predict quantitative effects of multiple functional groups. The mechanism(s) of retention seems more complex than can be explained simply by polarity or hydrophobicity. Since the molecular location of these functional moieties seems critical, retention and separation of sterols may involve specific structural configurations and hence specific interactions with the stationary phase material. The association we have drawn between polarity and HPLC elution may indeed be a secondary effect of another phenomenon. Future studies may unveil the true mechanism(s) of HPLC retention and separation, and allow for the construction of HPLC systems which will separate all isomeric combinations of sterols at the analytical level. The simplicity, rapidity, and reproducibility of these methods make the coupled technique very useful for investigating sterol metabolism. Application of this technique to analyzing putative sterol mutants, purifying sterols for auxotrophic feeding, and analyzing the metabolism of supplemented sterols in auxotrophs provides for significant advances in membrane physiology.

Chromatography, High Pressure Liquid↗
PubMed1985

Yeast sterols: yeast mutants as tools for the study of sterol metabolism.

Yeast mutants defective in ergosterol synthesis are valuable tools for investigating sterol metabolism. Both sterol mutants and sterol auxotrophs have been utilized in determining what sterol structural features are required for yeast cell viability. Both types of mutants can also be studied to ascertain how changes in sterol structure affect membrane properties. Other aspects of sterol metabolism, such as the specificity of sterol esterification, have been elucidated by the sterol auxotrophs. In broader applications, interrelationships between sterol metabolism and other cellular functions (e.g., heme metabolism) may also be examined with these mutants. By analyzing the lipid composition of the sterol mutants, on the other hand, much of the ergosterol biosynthetic pathway has been delineated. The unusual sterols of the mutants can also be obtained to develop assays for the enzymes involved in ergosterol synthesis. Thus, by utilizing mutants, the simple eukaryotic system of yeast may be extended to explore the entire field of sterol metabolism and its relationship to cellular physiology.

Chromatography, High Pressure Liquid↗
PubMed1985

Recovery of Saccharomyces cerevisiae from ethanol-induced growth inhibition.

Ethanol caused altered mobility of the lipophilic probe 1,6-diphenyl-1,3,5-hexatriene in plasma membrane preparations of Saccharomyces cerevisiae. Because lipids had been shown to protect yeast cells against ethanol toxicity, sterols, fatty acids, proteins, and combinations of these were tested; however, protection from growth inhibition was not seen. Ethanol-induced, prolonged lag periods and diminished growth rates in S. cerevisiae were reduced by an autoconditioning of the medium by the inoculum.

Adaptation, Physiological↗
PubMed1985

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.

5-Aminolevulinate Synthetase↗
PubMed1984

Sterol methylation in Saccharomyces cerevisiae.

Various nystatin-resistant mutants defective in S-adenosylmethionine: delta 24-sterol-C-methyltransferase (EC 2.1.1.41) were shown to possess alleles of the same gene, erg6. The genetic map location of erg6 was shown to be close to trp1 on chromosome 4. Despite the single locus for erg6, S-adenosylmethionine: delta 24-sterol-C-methyltransferase enzyme activity was found in three separate fractions: mitochondria, microsomes, and the "floating lipid layer." The amount of activity in each fraction could be manipulated by assay conditions. The lipids and lipid synthesis of mutants of Saccharomyces cerevisiae defective in the delta 24-sterol-C-methyltransferase were compared with a C5(6) desaturase mutant and parental wild types. No ergosterol (C28 sterol) could be detected in whole-cell sterol extracts of the erg6 mutants, the limits of detection being less than 10(-11) mol of ergosterol per 10(8) cells. The distribution of accumulated sterols by these mutants varied with growth phase and between free and esterified fractions. The steryl ester concentrations of the mutants were eight times higher than those of the wild type from exponential growth samples. However, the concentration of the ester accumulated by the mutants was not as great in stationary-phase cells. Whereas the head group phospholipid composition was the same between parental and mutant strains, strain-dependent changes in fatty acids were observed, most notably a 40% increase in the oleic acid content of phosphatidylethanolamine of one erg6 mutant, JR5.

Alleles↗
PubMed1983

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

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

Cholestanols↗