Influence of sterol structure on yeast plasma membrane properties

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Biochimica Et Biophysica Acta-biomembranes, 2011

The plasma membrane (PM) is a main site of injury during osmotic perturbation. Sterols, major lipids of the PM structure in eukaryotes, are thought to play a role in ensuring the stability of the lipid bilayer during physicochemical perturbations. Here, we investigated the relationship between the nature of PM sterols and resistance of the yeast Saccharomyces cerevisiae to hyperosmotic treatment. We compared the responses to osmotic dehydration (viability, sterol quantification, ultrastructure, cell volume, and membrane permeability) in the wild-type (WT) strain and the ergosterol mutant erg6Δ strain. Our main results suggest that the nature of membrane sterols governs the mechanical behavior of the PM during hyperosmotic perturbation. The mutant strain, which accumulates ergosterol precursors, was more sensitive to osmotic fluctuations than the WT, which accumulates ergosterol. The hypersensitivity of erg6Δ was linked to modifications of the membrane properties, such as stretching resistance and deformation, which led to PM permeabilization during the volume variation during the dehydration–rehydration cycles. Anaerobic growth of erg6Δ strain with ergosterol supplementation restored resistance to osmotic treatment. These results suggest a relationship between hydric stress resistance and the nature of PM sterols. We discuss this relationship in the context of the evolution of the ergosterol biosynthetic pathway.► Yeast mutant in sterol synthetic pathway (erg6Δ) is sensitive to hydric stress. ► Sterol nature affects plasma membrane integrity during hydric perturbation. ► Sterol structure influences plasma membrane deformation and resistance to stretching. ► Ergosterol complementation restores osmotic resistance in a sensitive mutant (erg6Δ). ► Ergosterol is involved in survival of wild-type yeast during hydric fluctuations.

Biochimica et Biophysica Acta (BBA) - Biomembranes, 1995

Fatty acyl esters of the yeast specifc sterol, ergosterol, are exclusively stored in lipid particles. Under conditions of sterol deficiency, e.g., in the presence of terbinafine, an inhibitor of fungal squalene epoxidase, steryl esters are hydrolyzed, and sterols are set free for membrane formation. Lipid particles do not contain steryl-ester hydrolase activity themselves; the highest specific activity of this enzyme is found in the plasma membrane. Therefore, steryl esters have to be exported from lipid particles to their site of hydrolytic cleavage. This process of translocation and :metabolic conversion was studied in vivo. Addition of nocodazole to terbinafine-treated cells did not disturb the mobilization of steryl esters, indicating that this process is not mediated by microtubuli-dependent vesicle flux. Under the influence of inhibitors of cellular energy production (azide and fluoride) and protein biosynthesis (cycloheximide) mobilization of steryl esters came to an halt. These results support the view that ongoing membrane proliferation may be a driving force for the release of sterols from steryl esters of lipid particles.

Biochimica et Biophysica Acta (BBA) - Biomembranes, 2004

In the yeast Saccharomyces cerevisiae, three enzymes of the sterol biosynthetic pathway, namely Erg1p, Erg6p and Erg7p, are located in lipid particles. Whereas Erg1p (squalene epoxidase) is also present in the endoplasmic reticulum (ER) to a significant amount, only traces of Erg6p (sterol C-24 methyltransferase) and Erg7p (lanosterol synthase) are found in the ER. We have chosen these three Erg-proteins as typical representatives of lipid particle proteins to study targeting to their destination. Lipid particle proteins do not contain obvious targeting motifs, but the only common structural feature is the presence of one or two hydrophobic domains near the C-termini. We constructed truncated versions of Erg1p, Erg6p and Erg7p to test the role of these hydrophobic domains in subcellular distribution. Our results demonstrate that lack of the hydrophobic domains prevents at least in part the association of the proteins with lipid particles and causes their retention to the ER. This result strongly supports the view that ER and lipid particles are related organelles.

Biophysical Chemistry, 2009

This paper is aimed at investigating sterol/phospholipid interactions in the exact proportion that occurs in fungi/mammalian cells. We have performed a thorough analysis of surface pressure (π)-area (A) isotherms with the Langmuir monolayer technique, complemented with Brewster angle microscopy (BAM) images. The following mixtures were analysed: cholesterol (Chol)-dipalmitoyl phosphatidylcholine (DPPC), Chol-dioleoyl phosphatidylcholine (DOPC), ergosterol (Erg)-DPPC, and Erg-DOPC. For each system, two different concentrations of the sterols were used, 13 and 30%, corresponding to the range of concentration found in various natural membranes. The obtained results show the existence of attractive interactions between phospholipids and cholesterol. Mixtures with ergosterol behave quite differently, i.e. either the interactions are repulsive (mixtures with DPPC) or the system is ideal (mixtures with DOPC). The obtained results have implications in the polyene antibiotics mode of action, i.e. the polyenes may interact easier with ergosterol, present in fungi cells, as compared to cholesterol-the main sterol of the mammalian cellular membranes.

European Journal of Biochemistry, 2000

Photodynamic treatment of the yeast Saccharomyces cerevisiae with the singlet oxygen sensitizer toluidine blue and visible light leads to rapid oxidation of ergosterol and accumulation of oxidized ergosterol derivatives in the plasma membrane. The predominant oxidation product accumulated was identified as 5a,6a-epoxy-(22E)-ergosta-8,22-dien-3b,7a-diol (8-DED). 9(11)-dehydroergosterol (DHE) was identified as a minor oxidation product. In heat inactivated cells ergosterol is photooxidized to ergosterol epidioxide (EEP) and DHE. Disrupted cell preparations of S. cerevisiae convert EEP to 8-DED, and this activity is abolished in a boiled control indicating the presence of a membrane associated enzyme with an EEP isomerase activity. Yeast selectively mobilizes ergosterol from the intracellular sterol ester pool to replenish the level of free ergosterol in the plasma membrane during singlet oxygen oxidation. The following reaction pathway is proposed: singlet oxygen-mediated oxidation of ergosterol leads to mainly the formation of EEP, which is enzymatically rearranged to 8-DED. Ergosterol 7-hydroperoxide, a known minor product of the reaction of singlet oxygen with ergosterol, is formed at a much lower rate and decomposes to give DHE. Changes of physical properties of the plasma membrane are induced by depletion of ergosterol and accumulation of polar derivatives. Subsequent permeation of photosensitizer through the plasma membrane into the cell leads to events including impairment of mitochondrial function and cell inactivation.

Frontiers in Cell and Developmental Biology, 2020

Here, biophysical properties of membranes enriched in three metabolically related sterols are analyzed both in vitro and in vivo. Unlike cholesterol and ergosterol, the common metabolic precursor zymosterol is unable to induce the formation of a liquid ordered (l o) phase in model lipid membranes and can easily accommodate in a gel phase. As a result, Zym has a marginal ability to modulate the passive membrane permeability of lipid vesicles with different compositions, contrary to cholesterol and ergosterol. Using fluorescence-lifetime imaging microscopy of an aminostyryl dye in living mammalian and yeast cells we established a close parallel between sterol-dependent membrane biophysical properties in vivo and in vitro. This approach unraveled fundamental differences in yeast and mammalian plasma membrane organization. It is often suggested that, in eukaryotes, areas that are sterolenriched are also rich in sphingolipids, constituting highly ordered membrane regions. Our results support that while cholesterol is able to interact with saturated lipids, ergosterol seems to interact preferentially with monounsaturated phosphatidylcholines. Taken together, we show that different eukaryotic kingdoms developed unique solutions for the formation of a sterol-rich plasma membrane, a common evolutionary trait that accounts for sterol structural diversity.

Journal of Biological Chemistry, 1981

Biochemistry, 1981

Stopped-flow kinetic measurements of the association of filipin with sterols in intact cells and isolated membranes of Mycoplasma capricolum were used to study the effects of varying the phospholipid composition and the sterol structure on sterol distribution in the membrane. The phospholipid composition and content of the membrane were varied by growing cells in an albumin-containing medium with cholesterol, palmitic and oleic acids, and various concentrations of exogenous phospholipids. The exogenous phospholipids (phosphatidylcholine, sphingomyelin, and phosphatidic acid) were incorporated up to levels of approximately 50% of the total membrane phospholipids but had no effect on the distribution of cholesterol between the two halves of the membrane bilayer. The sterol structure was varied by growing the R e c e n t studies have indicated that the distribution of cholesterol in the membrane of Mycoplasma capricolum can be examined on the basis of the rapid kinetics of filipin binding with cholesterol . In another sterol-requiring mycoplasma, M . gallisepticum, the estimate of the distribution of cholesterol between the two halves of the bilayer obtained by filipin binding was the same as that obtained by the kinetics of spontaneous cholesterol exchange with high-density lipoproteins . The filipin-binding approach was also used to examine the rate of transbilayer movement of cholesterol under physiological conditions, using an adapted strain of M. capricolum which grew on media containing low cholesterol concentrations . Since the sterol specificity of M . capricolum is relatively broad for review, see Smith (1979)l and since filipin binds to various sterols (Bittman & Fischkoff, 1972), we have used this approach to measure the transbilayer localization of a variety of sterols in the membrane of M . capricolum.

Chemistry and Physics of Lipids, 2002

Although the most exogenous lipids enter the cell via the LDL-receptor pathway, the mechanism(s) whereby lipids leave the lysosome for transport to intracellular sites are not clearly resolved. As shown herein, expression of sterol carrier protein-2 (SCP-2) in transfected L-cells altered lysosomal membrane lipid distribution, dynamics, and response to lipid transfer proteins. SCP-2 expression decreased the mass of cholesterol and lyso-bis-phosphatidic acid [LBPA], as well as the ratios of cholesterol/phospholipid and polyunsaturated/monounsaturated fatty acids esterified to lysosomal membrane phospholipids. Concomitantly, a fluorescent sterol transfer assay showed that SCP-2 expression decreased the initial rates of spontaneous and SCP-2-mediated sterol transfer 5.5-and 3.8-fold, respectively, from lysosomal membranes isolated from SCP-2 expressing cells as compared to controls. SCP-2, sphingomyelinase, low density lipoprotein, and high density lipoprotein directly enhanced the initial rates of sterol transfer from isolated lysosomal membranes by 50-, 12-, 4-, and 5-fold, respectively. In contrast, albumin and cholesterol esterase had no effect on lysosomal sterol transfer. Spontaneous sterol was very slow, t 1/2 \ 4 days, regardless of the source of the lysosomal membrane, while SCP-2 added in vitro induced formation of rapid and slowly transferable sterol pools in lysosomal membranes of control cells. In contrast, SCP-2 did not induce formation of a rapidly transferable sterol domain in lysosomal membranes isolated from SCP-2 expressing cells. These data suggest that SCP-2 expression selectively shifted the distribution of lipids (cholesterol, LBPA, esterified polyunsaturated fatty acids) away from lysosomal membranes. Furthermore, the cholesterol depleted lysosomal membrane isolated from SCP-2 expressing cells was resistant to additional direct action of SCP-2 to further enhance sterol transfer and induce rapidly transferable sterol pools in the lysosomal membrane.

Molecular Biology of the Cell, 1999

Sterols are major components of the plasma membrane, but their functions in this membrane are not well understood. We isolated a mutant defective in the internalization step of endocytosis in a gene (ERG2) encoding a C-8 sterol isomerase that acts in the late part of the ergosterol biosynthetic pathway. In the absence of Erg2p, yeast cells accumulate sterols structurally different from ergosterol, which is the major sterol in wild-type yeast. To investigate the structural requirements of ergosterol for endocytosis in more detail, several erg mutants (erg2⌬, erg6⌬, and erg2⌬erg6⌬) were made. Analysis of fluid phase and receptor-mediated endocytosis indicates that changes in the sterol composition lead to a defect in the internalization step. Vesicle formation and fusion along the secretory pathway were not strongly affected in the erg⌬ mutants. The severity of the endocytic defect correlates with changes in sterol structure and with the abundance of specific sterols in the erg⌬ mutants. Desaturation of the B ring of the sterol molecules is important for the internalization step.

PLOS ONE, 2015

We investigated the impact of the deletions of genes from the final steps in the biosynthesis of ergosterol (ERG6, ERG2, ERG3, ERG5, ERG4) on the physiological function of the Saccharomyces cerevisiae plasma membrane by a combination of biological tests and the diSC 3 (3) fluorescence assay. Most of the erg mutants were more sensitive than the wild type to salt stress or cationic drugs, their susceptibilities were proportional to the hyperpolarization of their plasma membranes. The different sterol composition of the plasma membrane played an important role in the short-term and long-term processes that accompanied the exposure of erg strains to a hyperosmotic stress (effect on cell size, pH homeostasis and survival of yeasts), as well as in the resistance of cells to antifungal drugs. The pleiotropic drug-sensitive phenotypes of erg strains were, to a large extent, a result of the reduced efficiency of the Pdr5 efflux pump, which was shown to be more sensitive to the sterol content of the plasma membrane than Snq2p. In summary, the erg4Δ and erg6Δ mutants exhibited the most compromised phenotypes. As Erg6p is not involved in the cholesterol biosynthetic pathway, it may become a target for a new generation of antifungal drugs.

Biochimica Et Biophysica Acta (bba) - Lipids and Lipid Metabolism, 1985

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.

Chemistry and Physics of Lipids, 2012

In this work, binary mixtures of phospholipid/ergosterol (erg) were studied using three fluorescent membrane probes. The phospholipid was either saturated (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPC) or monounsaturated (1-palmitoyl-2-dioleoyl-sn-glycero-3-phosphocholine, POPC) phosphatidylcholine, to evaluate the fluorescence properties of the probes in gel, liquid ordered (l o) and liquid disordered (l d) phases. The probes have been used previously to study cholesterol-enriched domains, but their photophysical properties in erg-enriched membranes have not been characterized. N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (NBD-DPPE) presents modest blue-shifts upon erg addition, and the changes in the fluorescence lifetime are mainly due to differences in the efficiency of its fluorescence dynamic self-quenching. However, the steady-state fluorescence anisotropy of NBD-DPPE presents well-defined values in each lipid phase. N-(lissamine rhodamine B sulfonyl)-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (Rhod-DOPE) presents a close to random distribution in erg-rich membranes. There are no appreciable spectral shifts and the steady-state fluorescence anisotropy presents complex behavior, as a result of different photophysical processes. The probe is mostly useful to label l d domains in yeast membranes. 4-(2-(6-(Dibutylamino)-2-naphthalenyl)ethenyl)-1-(3-sulfopropyl)-pyridinium (di-4-ANEPPS) is an electrochromic dye with excitation spectra largely insensitive to the presence of erg, but presenting a strong blue-shift of its emission with increasing concentrations of this sterol. Its partition coefficient is favorable to l o domains in POPC/erg mixtures. Although the fluorescence properties of di-4-ANEPPS are less sensitive to erg than to chol, in both cases the fluorescence lifetime responds monotonically to sterol mole fraction, becoming significantly longer in the presence of sterol as compared to pure POPC or DPPC bilayers. The probe displays a unique sensitivity to sterol-lipid interaction due to the influence of hydration and H-bonding patterns at the membrane/water interface on its fluorescence properties. This makes di-4-ANEPPS (and possibly similar probes) potentially useful in the study of erg-enriched domains in more complex lipid mixtures and in the membranes of living yeast cells.

Biochemical Journal, 2004

Morphological analysis of a conditional yeast mutant in acetyl-CoA carboxylase acc1 ts /mtr7, the rate-limiting enzyme of fatty acid synthesis, suggested that the synthesis of C 26 VLCFAs (verylong-chain fatty acids) is important for maintaining the structure and function of the nuclear membrane. To characterize this C 26 -dependent pathway in more detail, we have now examined cells that are blocked in pathways that require C 26 . In yeast, ceramide synthesis and remodelling of GPI (glycosylphosphatidylinositol)-anchors are two pathways that incorporate C 26 into lipids. Conditional mutants blocked in either ceramide synthesis or the synthesis of GPI anchors do not display the characteristic alterations of the nuclear envelope observed in acc1 ts , indicating that the synthesis of another C 26 -containing lipid may be affected in acc1 ts mutant cells. Lipid analysis of isolated nuclear membranes revealed the presence of a novel C 26 -substituted PI (phosphatidylinositol). This C 26 -PI accounts for approx. 1 % of all the PI species, and is present in both the nuclear and the plasma membrane. Remarkably, this C 26 -PI is the only C 26 -containing glycerophospholipid that is detectable in wild-type yeast, and the C 26 -substitution is highly specific for the sn − 1 position of the glycerol backbone. To characterize the biophysical properties of this lipid, it was chemically synthesized. In contrast to PIs with normal long-chain fatty acids (C 16 or C 18 ), the C 26 -PI greatly reduced the bilayer to hexagonal phase transition of liposomes composed of 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE). The biophysical properties of this lipid are thus consistent with a possible role in stabilizing highly curved membrane domains.