ESR determinations of membrane permeability in a yeast sterol mutant

Yeast sterol mutants were subjected to ESR analysis in an attempt to elucidate how altered sterol composition correlates with membrane permeability. The technique requires spin labeling the intact yeast cells with a small, water-soluble nitroxide probe (2,2,5,5 tetramethyl-3-pyrrolin-1-oxyl-3-carboxylic acid, PCA), suspending cells in a NiCl₂ solution, and measuring the extent of Ni²⁺ entry through the membrane by its magnetic dipolar line broadening effect on the PCA signal. The wild type, A184D, was found to be impermeable to Ni²⁺ during all growth phases while the sterol mutant $\text{erg}\text{6}\text{2}$ was readily permeable to Ni²⁺. Other sources of line broadening such as increased rotational correlation time and cell nonviability are shown to be negligible. Internal Ni²⁺ concentrations for $\text{erg}\text{6}\text{2}$ and kinetics of Ni²⁺ entry were determined.     

Flow cytometric determination of yeast sterol content

A method for the flow cytometric determination of the unesterfied 3β-hydroxysterol content in yeast populations by the fluorescence shifted macrolide nystatin in presented. Preliminary investigations of changing sterol content in aerobic and anaerobic batch cultivation revealed the technological usefullness of this information.     

Differences in crystal violet uptake and cation-induced death among yeast sterol mutants.

Yeast sterol mutants exposed to crystal violet demonstrated greater dye uptake than the wild type. In addition, exposure for 10 min to hypertonic cation solutions showed a greater decrease in cell viability for mutants than for wild type.     

Defective plasma membrane assembly in yeast secretory mutants.

Yeast mutants that are conditionally blocked at distinctive steps in secretion and export of cell surface proteins have been used to monitor assembly of integral plasma membrane proteins. Mutants blocked in transport from the endoplasmic reticulum (sec18), from the Golgi body (sec7 and sec14), and in transport of secretory vesicles (sec1) show dramatically reduced assembly of galactose and arginine permease activities. Simultaneous induction of galactose permease and alpha-galactosidase (a secreted glycoprotein) in sec mutant cells at the nonpermissive temperature (37 degrees C) shows that both activities accumulate and can be exported coordinately when cells are returned to the permissive temperature (24 degrees C) in the presence or absence of cycloheximide. Plasma membrane fractions isolated from sec mutant cells radiolabeled at 37 degrees C have been analyzed by two-dimensional sodium dodecyl sulfate-gel electrophoresis. Although most of the major protein species seen in plasma membranes from wild-type cells are not efficiently localized in sec18 or sec7, several of these proteins appear in plasma membranes from sec1 cells. These results may be explained by contamination of plasma membrane fractions with precursor vesicles that accumulate in sec1 cells. Alternatively, some proteins may branch off during transport along the secretory pathway and be inserted into the plasma membrane by a different mechanism.     

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.     

Influence of sterol structure on yeast plasma membrane properties

Fluorescence anisotropy measurements indicated that physical changes occured 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.     

Homoazasterol-mediated inhibition of yeast sterol biosynthesis.

A naturally occurring azasterol has been shown to inhibit sterol transmethylation in both in vitro and in vivo in the yeast Saccharomyces cerevisiae. The inhibition was competitive, with a calculated dissociation constant of 43 muM. The compound prevented the accumulation of ergosterol in aerobically adapting cells. Cultures forced to gain energy by respiration were found to be much more sensitive to growth inhibition by the azasterol than those cells fermenting glucose. The growth inhibition is reversible at low concentrations of the azasterol.     

A genomic screen for yeast vacuolar membrane ATPase mutants

V-ATPases acidify multiple organelles, and yeast mutants lacking V-ATPase activity exhibit a distinctive set of growth defects. To better understand the requirements for organelle acidification and the basis of these growth phenotypes, approximately 4700 yeast deletion mutants were screened for growth defects at pH 7.5 in 60 mm CaCl(2). In addition to 13 of 16 mutants lacking known V-ATPase subunits or assembly factors, 50 additional mutants were identified. Sixteen of these also grew poorly in nonfermentable carbon sources, like the known V-ATPase mutants, and were analyzed further. The cwh36Delta mutant exhibited the strongest phenotype; this mutation proved to disrupt a previously uncharacterized V-ATPase subunit. A small subset of the mutations implicated in vacuolar protein sorting, vps34Delta, vps15Delta, vps45Delta, and vps16Delta, caused both Vma- growth phenotypes and lower V-ATPase activity in isolated vacuoles, as did the shp1Delta mutation, implicated in both protein sorting and regulation of the Glc7p protein phosphatase. These proteins may regulate V-ATPase targeting and/or activity. Eight mutants showed a Vma- growth phenotype but no apparent defect in vacuolar acidification. Like V-ATPase-deficient mutants, most of these mutants rely on calcineurin for growth, particularly at high pH. A requirement for constitutive calcineurin activation may be the predominant physiological basis of the Vma- growth phenotype.     

AAA ATPases regulate membrane association of yeast oxysterol binding proteins and sterol metabolism

The yeast genome encodes seven oxysterol binding protein homologs, Osh1p-Osh7p, which have been implicated in regulating intracellular lipid and vesicular transport. Here, we show that both Osh6p and Osh7p interact with Vps4p, a member of the AAA (ATPases associated with a variety of cellular activities) family. The coiled-coil domain of Osh7p was found to interact with Vps4p in a yeast two-hybrid screen and the interaction between Osh7p and Vps4p appears to be regulated by ergosterol. Deletion of VPS4 induced a dramatic increase in the membrane-associated pools of Osh6p and Osh7p and also caused a decrease in sterol esterification, which was suppressed by overexpression of OSH7. Lastly, overexpression of the coiled-coil domain of Osh7p (Osh7pCC) resulted in a multivesicular body sorting defect, suggesting a dominant negative role of Osh7pCC possibly through inhibiting Vps4p function. Our data suggest that a common mechanism may exist for AAA proteins to regulate the membrane association of yeast OSBP proteins and that these two protein families may function together to control subcellular lipid transport.     

The ESR determination of protein alpha-helicity.

Experimental data are summarized on decay kinetics of uv-induced paramagnetic centers in globular proteins. The PC decay rate in the absence of oxygen in α helical regions is shown to differ always by about two orders from that in nonhelical regions of the polypeptide chain. This effect can be utilized to estimate the degree of α helicity in the dry state, in solution, and in the frozen solution of proteins.     

The effect of altered membrane sterol composition on the temperature dependence of yeast mitochondrial ATPase

The sterol content of cells of $\text{Saccharomyces cerevisiae}$ was manipulated by growing the organism anaerobically in a medium containing excess supplements of unsaturated fatty acids and a range of supplements of ergosterol. Anaerobic mitochondrial precursor structures were isolated whose membrane lipids contain the same fatty acid composition but whose sterol content varies from 7 to 105 mg/g mitochondrial protein. Arrhenius plots of the mitochondrial ATPase activity of the different preparations show a discontinuity with Arrhenius activation energies of about +40 and +80 KJ/mole, respectively, above and below the transition temperature. However, the temperature of the transition is markedly dependent on sterol composition, and increases by up to 17° as the sterol content of the mitochondria is progressively decreased. These results support the concepts that membrane lipid composition influences the activity of membrane-bound enzymes, and that sterols promote the gel to liquid phase transition in biological membranes.     

Location and regulation of early enzymes of sterol biosynthesis in yeast.

Acetoacetyl CoA thiolase and 3-hydroxy-3-methylglutaryl (HMG) CoA synthase were found almost entirely in the cytosol of Saccharomyces cerevisiae, whereas HMG CoA reductase was found almost entirely in mitochondria and further located in the matrix. Formation of all three enzymes was inhibited by cycloheximide, but not by chloramphenicol, indicating that they were synthesized in the cytosol. In anaerobically growing cells the levels of acetoacetyl CoA thiolase and HMG CoA synthase were decreased by ergosterol, whereas HMG CoA reductase levels were affected only slightly, suggesting that in yeast the enzymes responsible for synthesis of HMG CoA were regulated by ergosterol. Aerobically growing cells were essentially impermeable to ergosterol and cholesterol, whereas those growing anaerobically and requiring sterols were readily permeable. Mutants blocked in ergosterol formation were also permeable to sterols under aerobic conditions.     

Complementation of soluble phosphofructokinase activity in yeast mutants.

We describe here the genetic and biochemical analyses of two classes of mutations in the soluble phosphofructokinase (PFK I) of Saccharomyces cerevisiae: those leading to the loss of activity and those giving rise to a kinetically altered enzyme. Complementation and allele-testing between these two classes of mutants show that loss of enzyme activity in vitro can come about not only by mutations in the catalytic subunit but also in the regulatory subunit. Also, a mutation in the catalytic subunit can give rise to an enzyme altered in its kinetic properties in a manner phenomenologically similar to that caused by a mutation in the regulatory subunit. The results of the complementation studies in diploids suggest that, in spite of their distinct functions, both the subunits are essential for activity to be detected in vitro. This is confirmed by the reconstitution of an active PFK I enzyme by mixing cell-free extracts of two complementing parents, each of which lacks the enzyme activity. PFK activity appears in the mixture, reaching a maximum value of 60-100% of that of the diploid in 15-30 min at 24 degrees C. Unlike the catalytic subunit which exists in various multimeric states in cell-free extracts of the mutant bearing only this subunit, the regulatory subunit exists largely as a monomer in a mutant devoid of the catalytic subunit. The reconstituted enzyme, however, is indistinguishable from that of the wild type, as analysed by sedimentation studies and Western blot analysis, demonstrating that only the heteromeric complex of the two subunits is active, while neither of the individual subunits displays activity in vitro.     

Mechanisms of sterol uptake and transport in yeast

Sterols are essential lipid components of eukaryotic membranes. Here we summarize recent advances in understanding how sterols are transported between different membranes. Baker's yeast is a particularly attractive organism to dissect this lipid transport pathway, because cells can synthesize their own major sterol, ergosterol, in the membrane of the endoplasmic reticulum from where it is then transported to the plasma membrane. However, Saccharomyces cerevisiae is also a facultative anaerobic organism, which becomes sterol auxotroph in the absence of oxygen. Under these conditions, cells take up sterol from the environment and transport the lipid back into the membrane of the endoplasmic reticulum, where the free sterol becomes esterified and is then stored in lipid droplets. Steryl ester formation is thus a reliable readout to assess the back-transport of exogenously provided sterols from the plasma membrane to the endoplasmic reticulum. Structure/function analysis has revealed that the bulk membrane function of the fungal ergosterol can be provided by structurally related sterols, including the mammalian cholesterol. Foreign sterols, however, are subject to a lipid quality control cycle in which the sterol is reversibly acetylated. Because acetylated sterols are efficiently excreted from cells, the substrate specificity of the deacetylating enzymes determines which sterols are retained. Membrane-bound acetylated sterols are excreted by the secretory pathway, more soluble acetylated sterol derivatives such as the steroid precursor pregnenolone, on the other hand, are excreted by a pathway that is independent of vesicle formation and fusion. Further analysis of this lipid quality control cycle is likely to reveal novel insight into the mechanisms that ensure sterol homeostasis in eukaryotic cells. Article from a special issue on Steroids and Microorganisms.     

The effects of sterol mutants of the yeast Saccharomyces cerevisiae on the outcome of competition between Drosophila melanogaster and D. simulans

The effect of different yeast variants of the yeast Saccharomyces cerevisiae on the outcome of competition between Drosophila simulans and D. melanogaster was investigated. In all experiments differential birth rate was the effective mode of species competition. Addition of live yeast and especially mixtures of yeast strains improved the competition situation of a species, but could not prevent the extinction.     

Rescuing yeast mutants with human genes.

The fission yeast Schizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae have, in addition to being extensively studied themselves, both been utilized for the last quarter century as experimental systems for the isolation of genes from other organisms. Mutations conferring growth defects in either of the two yeast strains have frequently been complemented by expression of cDNA libraries from heterologous species, often human. Many successful experiments have utilized available yeast mutations to allow successful complementation by a human gene, which can thus be deduced to have the same, or an overlapping function as the mutated yeast gene. However complementation in yeast has also been used with success to study two fields, apoptosis and steroid receptor signalling, which, at first glance, seem to be foreign to the yeast life cycle.