The viscosity and lipid composition of the plasma membrane of multiple drug resistant and sensitive yeast strains.

Four different plasma membrane preparations were isolated from multiple drug resistant and sensitive isolates of two isogenic groups of Saccharomyces cerevisiae strains: zymolyase ghosts, concanavalin A ghosts, pH 4 nonaggregated vesicles, and sucrose-gradient purified vesicles. The viscosities of these preparations were determined by the use of a fluorescence polarization technique with 1,6-diphenyl-1,3,5-hexatriene. The viscosities of all four membrane preparations within an isogenic set were the same for resistant and sensitive strains. A comparison of the viscosity of zymolyase ghost liposomes showed that zymolyase ghost (glyco) proteins of resistant and sensitive strains had the same effect on viscosity. There was no difference between resistant and sensitive isolates in the mole concentration of the following lipid classes extracted from zymolyase ghosts: phospholipid, sterol, sterol ester, triglyceride, diglyceride, and free fatty acid. The fatty acid distribution of esterified and free fatty acids and the distribution of nine phospholipids was the same in zymolyase ghosts from sensitive and resistant strains. It was concluded that multiple drug resistance does not result from an alteration in plasma membrane viscosity or lipid composition.     

Sterol carrier protein-2 expression alters plasma membrane lipid distribution and cholesterol dynamics

Although sterol carrier protein-2 (SCP-2) binds, transfers, and/or enhances the metabolism of many membrane lipid species (fatty acids, cholesterol, phospholipids), it is not known if SCP-2 expression actually alters the membrane distribution of lipids in living cells or tissues. As shown herein for the first time, expression of SCP-2 in transfected L-cell fibroblasts reduced the plasma membrane levels of lipid species known to traffic through the HDL-receptor-mediated efflux pathway: cholesterol, cholesteryl esters, and phospholipids. While the ratio of cholesterol/phospholipid in plasma membranes of intact cells was not changed by SCP-2 expression, phosphatidylinositol, a molecule important to intracellular signaling and vesicular trafficking, and anionic phospholipids were selectively retained. Only modest alterations in plasma membrane phospholipid percent fatty acid composition but no overall change in the proportion of saturated, unsaturated, monounsaturated, or polyunsaturated fatty acids were observed. The reduced plasma membrane content of cholesterol was not due to SCP-2 inhibition of sterol transfer from the lysosomes to the plasma membranes. SCP-2 dramatically enhanced sterol transfer from isolated lysosomal membranes to plasma membranes by eliciting detectable sterol transfer within 30 s, decreasing the t(1/2) for sterol transfer 364-fold from >4 days to 7-15 min, and inducing formation of rapidly transferable sterol domains. In summary, data obtained with intact transfected cells and in vitro sterol transfer assays showed that SCP-2 expression (i) selectively modulated plasma membrane lipid composition and (ii) decreased the plasma membrane content cholesterol, an effect potentially due to more rapid SCP-2-mediated cholesterol transfer from versus to the plasma membrane.     

Regulation of transbilayer distribution of a fluorescent sterol in tumor cell plasma membranes

The lipid composition and transbilayer distribution of plasma membrane isolated from primary tumor (L-929, LM, A-9 and C3H) and nine metastatic cell lines cultured under identical conditions was examined. Cultured primary tumor and metastatic cells differed two-fold in sterol/phospholipid molar ratios. There was a direct correlation between plasma membrane anionic phospholipid (phosphatidylinositol and phosphatidylserine) content and plasma membrane sterol/phospholipid ratio. This finding may bear on the possible link between oncogenes and inositol lipids. The fluorescent sterol, dehydroergosterol, was incorporated into primary tumor and metastatic cell lines. Selective quenching of outer monolayer fluorescence by covalently linked trinitrophenyl groups demonstrated an asymmetric transbilayer distribution of sterol in the plasma membranes. The inner monolayer of the plasma membranes from both cultured primary and metastatic tumor cells was enriched in sterol as compared with the outer monolayer. Consistent with this, the inner monolayer was distinctly more rigid as determined by the limiting anisotropy of 1,6-diphenyl-1,3,5-hexatriene. Dehydroergosterol fluorescence was temperature dependent and sensitive to lateral phase separations in phosphatidylcholine vesicles and in LM cell plasma membranes. Dehydroergosterol detected phase separations near 24 degrees C in the outer monolayer and at 21 degrees C and 37 degrees C in the inner monolayer of LM plasma membranes. Yet, no change in transbilayer sterol distribution was detected in ascending or descending temperature scans between 4 and 45 degrees C. Alterations in plasma membrane phospholipid polar head group composition by choline analogues (N,N-dimethylethanolamine, N-methylethanolamine, and ethanolamine) also did not perturb transbilayer sterol asymmetry. Treatment with phenobarbital or prilocaine, drugs that selectively fluidize the outer and inner monolayer of LM plasma membranes, respectively, did not change dehydroergosterol transbilayer distribution.     

Relationship between ethanol tolerance, lipid composition and plasma membrane fluidity in Saccharomyces cerevisiae and Kloeckera apiculata

Abstract The lipid composition of a strain of each of two yeasts, Saccharomyces csrevisiae and Kloeckera apiculata , with different ethanol tolerances, was determined for cells grown with or without added ethanol. An increase in the proportion of ergosterol, unsaturated fatty acid levels and the maintenance of phospholipid biosynthesis seemed to be responsible for ethanol tolerance. The association of ethanol tolerance of yeast cells with plasma membrane fluidity, measured by fluorescence anisotropy, is discussed. We propose that an increase in plasma membrane fluidity may be correlated with a decrease in the sterol: phospholipid and sterol: protein ratios and an increase in unsaturation index.     

Corresponding changes in kynurenine hydroxylase activity, membrane fluidity, and sterol composition in Saccharomyces cerevisiae mitochondria.

The effect of sterol composition on the properties of the mitochondrial membrane of Saccharomyces cerevisiae was investigated. The physical state of mitochondrial membranes from wild-type strains and sterol mutants was compared, using a fluorescence polarization technique with 1,6-diphenyl-1,3-5-hexatriene. Changes in the rate of depolarization of the probe molecule as a function of temperature suggest the occurrence of a phase transition in the mitochondrial membranes isolated from the sterol mutants but not in the membranes isolated from the wild types. Arrhenius kinetics of the mitochondrial membrane-bound enzyme L-kynurenine-3-hydroxylase exhibited changes in activation energy at temperatures similar to those observed in the fluorescence polarization study. The ratio of mitochondrial sterol to phospholipid and the phospholipid fatty acid composition of the organisms were characterized.     

Lipid composition of a plasma membrane enriched fraction of maize roots

The lipid composition of a plasma membrane enriched fraction isolated from corn (Zea mays) roots was examined. On a wt basis, the lipid: protein ratio was 1.11. Phospholipids comprised 60% of total lipids with the major phospholipids being phosphatidylcholine (62%) and phosphatidylethanolamine (21%). Free sterol was the major neutral lipid. The sterol:phospholipid molar ratio was 0.31. The fatty acid composition of the membrane was predominantly linoleic (60%) and palmitic (30%).     

Effect of membrane sterol content on the susceptibility of phospholipids to phospholipase A2

The effects of membrane sterol level on the susceptibility of LM cell plasma membranes to exogenous phospholipases A2 has been investigated. Isolated plasma membranes, containing normal or decreased sterol content, were prepared from mutant LM cell sterol auxotrophs. beta-Bungarotoxin-catalyzed hydrolysis of both endogenous phospholipids and phospholipids introduced into the membranes with beef liver phospholipid exchange proteins was monitored. In both cases, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were degraded at similar rates in normal membranes, while PC hydrolysis was specifically accelerated in sterol-depleted membranes. Additional data suggest that this preferential hydrolysis of PC is not a consequence of the phospholipid head group specificity of the phospholipase, nor of a difference in the accessibility of PC versus PE to the enzyme. Analysis of the reaction products formed during treatment of isolated membranes with phospholipase A2 showed almost no accumulation of lysophospholipids. This was found to be due to highly active lysophospholipase(s), present in LM cell plasma membranes, acting on the lysophospholipids formed by phospholipase A2 action. A soluble phospholipase A2 was partially purified from LM cells and found to behave as beta-bungarotoxin with regard to membrane sterol content. These results demonstrate that the nature of phospholipid hydrolysis, catalyzed by phospholipase A2, can be significantly affected by membrane lipid composition.     

Effect of Milk Components on IgA Production in Peyer’s Patch Cell Cultures from Mouse

The lipid composition of some commercial bakers’ yeasts having different freeze-sensitivity in frozen dough was investigated to clarify the correlation between their lipid composition and freeze-tolerance. The total lipid content including neutral lipid, free fatty acid, sterol, and phospholipid ranged between 23.0 to 32.2 mg/100 mg protein of the yeasts tested. Phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine were the main phospholipids found in all yeast strains, but no distinct difference in these components between freeze-tolerant and freeze-sensitive strains was observed. Palmitoleic (C16:l), oleic (C18:l), palmitic (16:0), and stearic (CI8:0) acids were the major fatty acids present in total lipid and phospholipid, and unsaturation indices of fatty acid in these lipid components were almost equal by the strains. The molar ratios of sterol to phospholipid of freeze-sensitive strains were higher than those of freeze-tolerant strains. The difference in the sterol-pho-spholipid ratio that influences the fluidity of plasma membranes in yeast cells was supposed to reflect the difference in freeze-sensitivity of bakers’ yeast.     

Fluidity and lipid composition of oat and rye shoot plasma membrane: effect of sterol perturbation by xenobiotics

Oat and rye plants were treated with either tetcyclacis (an experimental plant growth regulator), nuarimol (a fungicide) or gamma-ketotriazole (an experimental herbicide). These treatments reduced shoot growth and changed the lipid composition of the shoot plasma membranes. In oat, both tetcyclacis and nuarimol treatments increased plasma membrane cholesterol and increased the phosphatidylethanolamine/phosphatidylcholine (PE/PC) ratio, whereas gamma-ketotriazole treatment reduced cholesterol and the PE/PC ratio. In rye, all treatments reduced the PE/PC ratio. Generally, the sterol/phospholipid ratio was less in oat than in rye but the cholesterol/phospholipid ratio was greater. With all treatments in oat and rye, increases were observed in unsaturation of the phospholipid acyl chains. The fluidity of membranes was measured by steady-state fluorescence polarisation of the probe diphenylhexatriene; oat membranes were more fluid than rye. Membrane fluidity was greater in plasma membranes from plants treated with the xenobiotics than the controls. The results are discussed in the context of the effect of plasma membrane lipid composition on membrane fluidity, and it is concluded that there appears to be no overall simple relationship between membrane lipid composition and fluidity that holds for all treatments in both species.     

Correlation of tumor metastasis with sterol carrier protein and plasma membrane sterol levels

Squalene and sterol carrier protein (SCP) levels and sterol/phospholipid molar ratios of whole cells and plasma membranes were measured in cultured primary tumor and metastatic cell lines. SCP is abundant in all cell lines. However, metastatic lines have significantly lower SCP levels and plasma membrane sterol/phospholipid ratios than do primary lines. The results indicate that extremely malignant, metastatic cells are unable to produce or maintain adequate levels of both SCP and plasma membrane sterols when grown in lipoprotein deficient media. This defect, in vivo, probably causes excess uptake of SCP and lipid.     

Hydrogen fluoride effects on plasma membrane composition and ATPase activity in needles of white pine (Pinus strobus) seedlings pretreated with 12 h photoperiod

 Eastern white pine (Pinus strobus L.) seedlings were pretreated with 12 h photoperiod to induce dormancy. Dormant plants were fumigated with 0.5 ppb (0.4 μg m^–3) or 2.0 ppb (1.6 μg m^–3) hydrogen fluoride (HF) for 2 – 28 days. Plasma membranes were isolated from needles of treated and control seedlings to determine their chemical composition and ATPase activity. For all analyses, only those plants which did not show needle necrosis were selected. The amount of plasma membrane phospholipid expressed on a plasma membrane protein basis was higher after 2 days in the 0.5 ppb HF treatment as compared to controls. After 2 days of 2.0 ppb HF treatment as well as after 8 and 28 days of both HF treatments phospholipid to protein ratios in fluoride treated seedlings were lower as compared to control levels. A decrease in sterol levels could be observed after 2 days in both HF treatments. A large increase in the ratio of sterols to proteins was observed in plasma membranes of eastern white pine seedlings treated with 0.5 ppb HF for 28 days. Increased sterol to phospholipid ratios were observed after 8 and 28 days in 0.5 ppb and after 2 and 8 days of 2.0 ppb HF treatment. A decrease in ATPase activity was observed after 8 days with both fluoride treatments. Drastic increase of ATPase activity was observed after 28 days of HF treated plants. Observed changes of sterol and phospholipid levels after only 2 days of fumigation suggest early fluoride effects on plasma membrane composition during plant dormancy. Received: 25 October 1995 / Accepted: 24 May 1996     

Altered membrane structure in transfected mouse L-cell fibroblasts expressing rat liver fatty acid-binding protein

Mouse L cell fibroblasts were transfected with cloned cDNA encoding rat liver fatty acid binding protein (L-FABP) also known as sterol carrier protein. Stable transfectant cell lines were selected and expression of L-FABP determined using Western blot analysis. The nontransfected controls and low expression cells did not differ significantly in any of the properties examined. All cell lines showed similar doubling times but cells expressing high levels of L-FABP attained 2-fold higher cell saturation density and differed significantly in their lipid metabolism as indicated by 1) higher cholesterol ester and phospholipid content, and 2) decreased sterol/phospholipid ratio. The observed changes in the lipid composition predicted a lower degree of membrane-lipid order (higher fluidity) in the plasma membranes of cells expressing high levels of L-FABP. Therefore, fluorescent molecule, 1,6-diphenyl-1,3,5-hexatriene, and multifrequency (1-250 MHz) phase and modulation fluorometry were used to probe the effect of L-FABP expression on membrane structure. Steady-state polarization and limiting anisotropy of diphenylhexatriene were significantly lower in the isolated plasma membrane vesicles from the high expression clones. The observed changes in L-cells as a result of de novo expression of L-FABP are consistent with the ability of this protein to bind sterols and fatty acids, stimulate sterol esterification, and stimulate phospholipid biosynthesis. This evidence is supportive of a physiologic role for L-FABP in modulating cellular lipid metabolism and membrane structure.     

Modulation of the lipid composition of boar sperm plasma membranes during an acrosome reaction in vitro

An in vitro acrosome-like reaction was induced in spermatozoa from the boar cauda epididymis by incubation in Tyrode's solution containing 1 mg/ml fatty acid-free bovine serum albumin. Plasma membranes were isolated from the spermatozoa at different times during the incubation and analyzed for their lipid composition. The total lipid, phospholipid, and glycolipid content of the membranes did not change during the acrosome-like reaction, whereas the amount of diacylglycerols and free fatty acids increased. Within the phospholipid class, a decrease of the inositol phospholipid and and sphingomyelin content was observed, whereas the other phospholipids of the plasma membranes did not decrease significantly after 2 h of incubation. Changes in the sterol composition of the membranes were also observed. The onset of the lipid changes was correlated with the uptake of extracellular calcium by the spermatozoa. These results for the lipid changes in isolated sperm plasma membranes during an in vitro acrosome reaction provide the first direct evidence that a modulation of the plasma membrane lipid composition is involved in an acrosome-like reaction of mammalian spermatozoa.     

Effects of paclobutrazol and chilling temperatures on lipids, antioxidants and ATPase activity of plasma membrane isolated from green bell pepper fruits

The effects of paclobutrazol treatment on plasma membrane lipid composition and ATPase activity of bell pepper fruit ( Capsicum annuum ) subjected to chilling temperatures were assessed. Application of the growth regulator paclobutrazol affected plant growth and fruit morphology. The plants were more compact and the fruits were less elongated than control fruits. There was about 60% more plasma membrane on a fresh weight basis from treated fruits. At harvest there was no difference in sterol to phospholipid ratio, or in phospholipid fatty acid composition of control compared with paclobutrazol treated fruit. However, plasma membrane ATPase acitivity of treated fruit was two times higher than that of control fruit. After storage at chilling temperature (2°C), the control fruit developed more chilling iniury, and had greater weight loss and a higher rate of K⁺ leakage than paclobulrazol treated fruit. Plasma membrane phospholipid content decreased and saturation of phospholipid fatty acids was higher than in control fruit. These two changes were largely absent in plasma membrane from treated fruit. At harvest antioxidant levels in the plasma membrane of paclobutrazol treated peppers were higher than in those of controls and changed little during storage, whereas levels in control fruit plasma membrane decreased 66%. ATPase activity increased and then decreased in control fruit held at low temperature, whereas in treated fruit activity was constant. The protective effect of paclobutrazol against chilling injury of pepper fruit may result from a combination of its effect on fruit morphology, and protection of the lipids against oxidative stress.     

Distribution of membranes, especially of plasma-membrane fragments, during zonal centrifugations of homogenates from glucose-repressed Saccharomyces Cerevisiae.

1. The distributions of several enzymes and other marker components were examined after zonal centrifugations of whole homogenates from glucose-repressed Saccharomyces cerevisiae on sucrose and iso-osmotic Ficoll, and the composition and morphology of the fractions were investigated. 2. After high-speed zonal centrifugation most of the protein, acid and alkaline phosphatases, alkaline pyrophosphatase, adenosine monophosphatase, beta-fructofuranosidase, alpha-mannosidase, NADPH-cytochrome c oxidoreductase and an appreciable amount of phospholipid and sterol were non-sedimentable, i.e. were at densities below 1.09 (g/cm3). Most of the RNA was at p=1.06-1.08 in Ficoll and at p=1.09-1.11 in sucrose. 3. The bulk of the Mg2+-dependent adenosine triphosphatase (Mg-ATPase) was coincident with the main peak of phospholipid and sterol, at median density 1.10, which was also rich in smooth-membrane vesicles. In Ficoll, a minor peak of phospholipid and sterol at p-1.12-1.15 contained a smaller part of the oligomycin-insensitive Mg-ATPase and heavy membrane fragments. In sucrose, several minor peaks of Mg-ATPase were in the mitochondrial density range, and a peak of oligomycin-insensitive Mg-ATPase coincident with a minor peak of phospholipid and sterol at around p-1.25 contained heavy membrane fragments of high carbohydrate content, especially mannose. 4. Further purification of the oligomycin-insensitive Mg-ATPase containing membrane preparations was performed on Urografin gradients. 5. It is argued that the oligomycin-insensitive Mg-ATPase containing membranes are fragments of the plasma membrane, but have different densities because they contain different amounts of glycoprotein particles.     

Tolerance to NaCl induces changes in plasma membrane lipid composition,fluidity and H+-ATPase activity of tomato calli

Two tomato ( Lycopersicon esculentum Mill. cv. Pera) callus lines tolerant to NaCl were obtained by successive subcultures of NaCl-sensitive calli in 50 and 100 m M NaCl-supplemented medium. Growth and ion content, as well as plasma membrane lipid composition, fluidity and H⁺-ATPase (EC 3.6.1.35) activity, were studied in both NaCl-sensitive and NaCl-tolerant calli. Although calli tolerant to 100 m M NaCl exhibited a reduced growth relative to calli sensitive to NaCl or tolerant to 50 m M NaCl, growth of calli tolerant to 100 m M NaCl was higher than that of NaCl-sensitive calli grown for one subculture in 100 m M NaCl. Growth in the presence of 100 m M NaCl provoked an increase of Na⁺ and Cl⁻ content, but no significant changes in K⁺ and Ca²⁺. As compared with NaCl-sensitive and 50 m M NaCl-tolerant calli, plasma membrane vesicles isolated from calli tolerant to 100 m M NaCl exhibited a higher phospholipid and sterol content as well as a lower phospholipid/free sterol ratio and a lower double bond index (DBI) of phospholipid fatty acids. The changes in plasma membrane lipid composition were correlated with a decrease of plasma membrane fluidity in calli tolerant to 100 m M NaCl, as indicated by fluorimetric studies using diphenylhexatriene (DPH) as probe. Plasma membrane-enriched vesicles isolated from calli tolerant to 100 m M NaCl showed lower ATP hydrolysis and ATP-dependent H⁺-pumping activities, as well as a lower passive permeability to H⁺ than plasma membrane from NaCl-sensitive and 50 m M NaCl-tolerant calli. The involvement of the changes in plasma membrane lipid content and composition, fluidity and H⁺-ATPase activity in salt tolerance of tomato calli is discussed.     

Phospholipid flippases and Sfk1 are essential for the retention of ergosterol in the plasma membrane

Sterols are important lipid components of the plasma membrane (PM) in eukaryotic cells, but it is unknown how the PM retains sterols at a high concentration. Phospholipids are asymmetrically distributed in the PM, and phospholipid flippases play an important role in generating this phospholipid asymmetry. Here, we provide evidence that phospholipid flippases are essential for retaining ergosterol in the PM of yeast. A mutant in three flippases, Dnf1-Lem3, Dnf2-Lem3, and Dnf3-Crf1, and a membrane protein, Sfk1, showed a severe growth defect. We recently identified Sfk1 as a PM protein involved in phospholipid asymmetry. The PM of this mutant showed high permeability and low density. Staining with the sterol probe filipin and the expression of a sterol biosensor revealed that ergosterol was not retained in the PM. Instead, ergosterol accumulated in an esterified form in lipid droplets. We propose that ergosterol is retained in the PM by the asymmetrical distribution of phospholipids and the action of Sfk1. Once phospholipid asymmetry is severely disrupted, sterols might be exposed on the cytoplasmic leaflet of the PM and actively transported to the endoplasmic reticulum by sterol transfer proteins.     

Effect of active dry wine yeast storage upon viability and lipid composition

Alterations in the plasma membrane of Active Dry Wine Yeast (ADWY) produce deleterious effects upon yeast fermentation activity. The aim of this study was to evaluate the changes in viability mediated by plasma membrane alterations induced by poor ADWY storage conditions. Yeast recovery was analyzed after growth in optimal medium. The effects upon vitality were measured by impedance variation. Cell membrane functionality was measured by anisotropy and cell lipid composition was also determined. The results showed a reduction in viability of up to 57–87% in the stored yeast. The storage effect increased saturated fatty acids, and reduced unsaturated fatty acids and phospholipid contents. The stored yeast recovery was related to membrane functionality and significant increases in unsaturated fatty acid, sterol and phospholipid concentrations. Good vitality was positively correlated to high unsaturated fatty acid, phosphatidylserine and phosphatidylethanolamine concentrations and negatively to high saturated fatty acid and phosphatidylcholine contents.     

Stress tolerance in the marsh plant Spartina patens: Impact of NaCl on growth and root plasma membrane lipid composition

The C‐4 salt marsh grass, Spartina patens , thrives in the upper portion of the marsh where soil salinities may be equal to coastal seawater. Spartina patens was grown in hydroponic culture in a greenhouse at 0, 340, and 510 m M NaCl, and measured for growth, tissue cation content, and root plasma membrane (PM) lipid composition. From 0 to 340 and 510 m M , the shoot growth decreased, but root growth was not affected. The Na⁺ content increased in both shoots and roots when plants were grown in salt, while the shoots had a decreased K⁺ content and the roots had a decreased Ca²⁺ content. Spartina patens root plasma membrane was isolated with an aqueous polymer two‐phase system. The purity of the plasma membrane was verified with cytochemical tests on membrane enzyme markers. Plasma membrane lipids were stable relative to the membrane protein content. Molar percentages of sterols (including free sterols) and phospholipid decreased with increasing salinity. However, glycolipid showed a statistically significant increase in the total lipid as salinity in the medium was increased from 0 to 510 m M . Even at a salinity of 510 m M , the plasma membrane sterol/phospholipid ratio was unaffected by NaCl. When the plants were grown in NaCl media, the plasma membrane had a decreased phosphatidylcholine (PC) and phosphatidylethanolamine (PE) content, but the PC/PE ratios were not affected. The plasma membrane molar percentage of sitosterol in total free sterol increased when plants were grown in salt media. The predominant membrane fatty acids were C11 and C14, and the major unsaturated one was C14:1. An increase in growth medium salinity resulted in a decreased root plasma membrane fluidity.     

The effects of cotyledon senescence on the composition and physical properties of membrane lipid

The phospholipid content of rough and smooth microsomal fractions from cotyledons of germinating bean declines as the tissue becomes senescent. Both types of membrane contain comparable proportions of three major phospholipids, phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol, which collectively comprise about 90% of the total. This proportionality does not change appreciably during senescence. Only small quantities of lysophosphatides were noted at all stages of senescence. The unsaturated:saturated fatty acid ratio for total extracted lipid declined only slightly in both membrane systems, but pronounced differences in this ratio were observed among the major phospholipids of the membranes. The most striking alteration in lipid composition with advancing senescence was an increase in the sterol:phospholipid ratio; this rose by about 50% for rough microsomes and 400% for smooth microsomes. For both types of membrane the patterns of change in this ratio correlated with previously reported changes in bulk lipid transition temperature, suggesting that the increase in sterol level may contribute to changes in phase behaviour of the membranes during senescence. Arrhenius plots of rotational correlation times for the electron spin label 2,2-dimethyl-5-dodecyl-5-methyloxazolidine-N-oxide (2N14) partitioned into the membrane lipid showed an increase in viscosity with advancing senescence and a corresponding increase in activation energy for both types of membrane. These changes in activation energy and viscosity correlated closely with the increase in sterol:phospholipid ratio. However, no phase transitions were detectable between temperatures of 2 and 55 degrees C despite the fact that transitions from a lipid-crystalline to gel state are detectable within this temperature range by wide angle X-ray diffraction.