Phosphatidylinositol phosphate, phosphatidylinositol bisphosphate, and the phosphoinositol sphingolipids are found in the plasma membrane and stimulate the plasma membrane H(+)-ATPase of Saccharomyces cerevisiae.

Several plasma membrane phospholipids have been studied for their ability to modulate the activity of the plasma membrane H(+)-ATPase of Saccharomyces cerevisiae. We show here that phosphatidylinositol phosphate (PIP), phosphatidylinositol bisphosphate (PIP2), and/or the phosphatidylinositol and PIP kinases are localized primarily in the plasma membrane. Previous in vivo studies with S. cerevisiae have shown that large, rapid, and reversible changes occur in the levels of PIP and PIP2 congruent with changes in cellular ATP levels. We demonstrate here that isolated plasma membranes exhibit the same changes in PIP and PIP2 content when they are supplied with or washed free of ATP. Using a mixed micellar assay we systematically studied the efficacy of the plasma membrane lipids in sustaining the activity of the plasma membrane H(+)-ATPase. We demonstrate for the first time that a number of plasma membrane glycerophospholipids effectively stimulate the ATPase, including PIP, PIP2, and cardiolipin. Phosphoinositol-containing sphingolipids, major components of the plasma membrane, are also shown to stimulate the ATPase at significantly lower levels than the glycerophospholipids and must also be considered as important effectors in vivo.     

Multiple lipid transport pathways to the plasma membrane in yeast

The plasma membrane of the yeast Saccharomyces cerevisiae is devoid of lipid-synthesizing enzymes, but contains all classes of bilayer-forming lipids. As the lipid composition of the plasma membrane does not match any of the intracellular membranes, specific trafficking of lipids from internal membranes, especially the endoplasmic reticulum and the Golgi, to the cell periphery is required. Although the secretory pathway is an obvious route to translocate glycerophospholipids, sphingolipids and sterols to the plasma membrane, experimental evidence for the role of this pathway in lipid transport is rare. Addressing this issue in a systematic way, we labeled temperature-sensitive secretory yeast mutants (sec mutants) with appropriate lipid precursors, isolated the plasma membranes at high purity and quantified labeled lipids of this compartment. Shifting sec mutants to the restrictive temperature reduced transport of both proteins and lipids to the plasma membrane, indicating that the latter compounds are also trafficked to the cell periphery through the protein secretory pathway. However, efficient sec blocks did not abrogate protein and lipid transport, suggesting that parallel pathway(s) for the translocation of membrane components to the plasma membrane of yeast must exist.     

Lipid raft-based membrane compartmentation of a plant transport protein expressed in Saccharomyces cerevisiae

The hexose-proton symporter HUP1 shows a spotty distribution in the plasma membrane of the green alga Chlorella kessleri. Chlorella cannot be transformed so far. To study the membrane localization of the HUP1 protein in detail, the symporter was fused to green fluorescent protein (GFP) and heterologously expressed in Saccharomyces cerevisiae and Schizosaccharomyces pombe. In these organisms, the HUP1 protein has previously been shown to be fully active. The GFP fusion protein was exclusively targeted to the plasma membranes of both types of fungal cells. In S. cerevisiae, it was distributed nonhomogenously and concentrated in spots resembling the patchy appearance observed previously for endogenous H(+) symporters. It is documented that the Chlorella protein colocalizes with yeast proteins that are concentrated in 300-nm raft-based membrane compartments. On the other hand, it is completely excluded from the raft compartment housing the yeast H(+)/ATPase. As judged by their solubilities in Triton X-100, the HUP1 protein extracted from Chlorella and the GFP fusion protein extracted from S. cerevisiae are detergent-resistant raft proteins. S. cerevisiae mutants lacking the typical raft lipids ergosterol and sphingolipids showed a homogenous distribution of HUP1-GFP within the plasma membrane. In an ergosterol synthesis (erg6) mutant, the rate of glucose uptake was reduced to less than one-third that of corresponding wild-type cells. In S. pombe, the sterol-rich plasma membrane domains can be stained in vivo with filipin. Chlorella HUP1-GFP accumulated exactly in these domains. Altogether, it is demonstrated here that a plant membrane protein has the property of being concentrated in specific raft-based membrane compartments and that the information for its raft association is retained between even distantly related organisms.     

Lipids of rabies virus and BHK-21 cell membranes.

The lipid composition of highly purified Flury strain of rabies virus (HEP) propagated in BHK-21 cells in a chemically defined medium was observed to be 6.7% neutral lipids, 15.8% phospholipids, and 1.5% glycolipids. In the virion, phosphatidylethanolamine, phosphatidylcholine, and sphingomyelin were the most abundant phospholipids, accounting for 90% of the total, and the molar ratio of cholesterol to phospholipid was 0.48. Uninfected BHK-21 cell membranes were obtained by nitrogen cavitation techniques and separated by density gradient centrifugation, and the membranes were assayed for purity using 5'-nucleotidase, cytochrome oxidase, and reduced nicotinamide adenine dinucleotide phosphate diaphorase activities. Lipids of the plasma membrane were enriched in cholesterol, phosphatidylcholine, and phosphatidylethanolamine. In contrast, membranes of the endoplasmic reticulum were enriched in phosphatidylcholine, but contained smaller amounts of phosphatidylethanolamine and sphingomyelin. Comparison of the fatty acyl chains of virus and membranes from uninfected cells revealed the virion to have the lowest ratio of C18:1 to C18:0 (1.771), compared with values of about 3.0 for the plasma membrane and endoplasmic reticulum. Total polyenoic fatty acids were enriched in the plasma membrane, whereas the virus contained higher amounts of total saturates than either of the two membrane preparations. Analysis of the polar and neutral lipid fractions as well as the acyl chain analysis suggests the virion has a lipid composition that is intermiediate to that of the plasma membrane and endoplasmic reticulum and is consistent with the view that numerous viral particles are synthesized de novo by not utilizing a preexisting membrane template. From the ratio of cholesterol to phospholipid of 0.48, we calculated that 1.92 X 10(5) molecules of lipid would cover 4.14 X 10(4) nm2 in the form of a bilayer. Considerations of the molecular dimensions of the rabies envelope (total surface area, 5 X 10(4) nm2) as a bilayer suggest that some penetration of lipids by envelope proteins (M and G) is necessary.     

A putative plant aminophospholipid flippase, the Arabidopsis P4 ATPase ALA1, localizes to the plasma membrane following association with a β-subunit

Plasma membranes in eukaryotic cells display asymmetric lipid distributions with aminophospholipids concentrated in the inner leaflet and sphingolipids in the outer leaflet. This unequal distribution of lipids between leaflets is, amongst several proposed functions, hypothesized to be a prerequisite for endocytosis. P4 ATPases, belonging to the P-type ATPase superfamily of pumps, are involved in establishing lipid asymmetry across plasma membranes, but P4 ATPases have not been identified in plant plasma membranes. Here we report that the plant P4 ATPase ALA1, which previously has been connected with cold tolerance of Arabidopsis thaliana, is targeted to the plasma membrane and does so following association in the endoplasmic reticulum with an ALIS protein β-subunit.     

Galactosyltransferase activity is restricted to the plasma membranes of equine and bovine sperm

beta 1, 4-Galactosyltransferase (GalTase) is localized to the plasma membrane of mouse sperm, in which it mediates the binding of sperm to glycoconjugate residues in the egg zona pellucida. In this study, the presence of subcellular distribution of sperm GalTase were determined in two other mammalian species that yield sufficient sperm for subcellular fractionation. Equine and bovine semen were collected, and the plasma membranes (PM), outer acrosomal membranes (OAM), and inner acrosomal membranes (IAM) were sequentially removed. The purities of the isolated membrane preparations were determined by transmission electron microscopy and found to be greater than or equal to 90%, 96%, and 98% for equine PM, OAM, and IAM, respectively, and greater than or equal to 80%, 94%, and 97% for bovine PM, OAM, and IAM, respectively. GalTase activity was assayed under optimal conditions in all membrane preparations and was preferentially localized to the isolated PM both in equine and in bovine spermatozoa. The selective localization of GalTase to the sperm PM in two other species suggest that it may serve as a generalized gamete receptor during initial sperm-egg binding in mammals.     

Sphingolipid topology and the dynamic organization and function of membrane proteins

When acquiring internal membranes and vesicular transport, eukaryotic cells started to synthesize sphingolipids and sterols. The physical differences between these and the glycerophospholipids must have enabled the cells to segregate lipids in the membrane plane. Localizing this event to the Golgi then allowed them to create membranes of different lipid composition, notably a thin, flexible ER membrane, consisting of glycerolipids, and a sturdy plasma membrane containing at least 50% sphingolipids and sterols. Besides sorting membrane proteins, in the course of evolution the simple sphingolipids obtained key positions in cellular physiology by developing specific interactions with (membrane) proteins involved in the execution and control of signaling. The few signaling sphingolipids in mammals must provide basic transmission principles that evolution has built upon for organizing the specific regulatory pathways tuned to the needs of the different cell types in the body.     

Role of inositol-containing sphingolipids in Saccharomyces cerevisiae during inositol starvation.

The in vitro lipid requirements of UDP-N-acetylglucosamine-dolichol phosphate N-acetylglucosamine-1-phosphotransferase for the inositol-containing sphingolipids from Saccharomyces cerevisiae were characterized in terms of concentration and specificity. The effects of combinations of lipids, especially phosphatidylinositol and the inositol-containing sphingolipids, were also tested on the transferase. Phosphatidylinositol and phosphatidylglycerol stimulated the enzyme 3.3- and 2.8-fold, respectively. The inositol-containing sphingolipids, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine did not stimulate the activity of the transferase. Phosphatidylcholine and phosphatidylethanolamine in combination with phosphatidylinositol had no effect on the transferase activity; however, the inositol-containing sphingolipids markedly inhibited the stimulation of the transferase by phosphatidylinositol. This inhibition by the sphingolipids was prevented if phosphatidylcholine, in addition to the other lipids, was present in the assay mixture. In addition, changes due to inositol starvation in the in vivo membrane lipid environment, i.e., phosphatidylinositol and the inositol-containing sphingolipids, were analyzed to determine whether they corresponded to the observed in vitro effects. Three hours after the beginning of inositol starvation, there were 9- and 14-fold reductions in the accumulation of phosphatidylinositol in membrane fractions IIA (vesicles) and IV (endoplasmic reticulum), respectively, although there was only a 6-fold reduction in membrane fraction I (plasma membrane). The accumulation of [14C]inositol into inositol-containing sphingolipids also reflected the differences in the cellular location of membranes.     

The plasma membrane of yeast acquires a novel heat-shock protein (hsp30) and displays a decline in proton-pumping ATPase levels in response to both heat shock and the entry to stationary phase.

Recent studies have revealed that the action of the proton-translocating ATPase of the plasma membrane of yeast is an important determinant of several stress tolerances and affects the capacity of cells to synthesise heat shock proteins in response to heat shock [Panaretou, B. & Piper, P. W. (1990) J. Gen. Microbiol. 136, 1763-1770; Coote, P. J., Cole, M. B. & Jones, M. V. (1991) J. Gen. Microbiol. 137, 1701-1708]. This study investigated the changes to the protein composition of the Saccharomyces cerevisiae plasma membrane that result from a heat shock to dividing cultures and the entry to stationary growth caused by carbon source limitation. Plasma membranes were prepared from exponential, heat-shocked and stationary yeast cultures. The proteins of these membrane preparations were then analysed by polyacrylamide gel electrophoresis and immunoblot measurement of ATPase levels. The protein composition of plasma membranes displayed two prominent changes in response to both heat shock and the entry to stationary phase: (a) a reduction in the level of the plasma membrane ATPase; and (b) the acquisition of a previously uncharacterised 30 kDa heat-shock protein (hsp30). The ATPase decline with heat shock probably exerts an important influence over the ability of the cell to maintain ATPase activity, and therefore intracellular pH, during extended periods of stress. Through in vivo pulse-labelling of plasma membrane proteins synthesised before and during heat shock, followed by subcellular fractionation, it was shown that hsp30 is the only protein induced by the yeast heat-shock response that substantially copurifies with plasma membranes. It might therefore exert a stress-protective function specifically at this membrane.     

Electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis of the lipid molecular species composition of yeast subcellular membranes reveals acyl chain-based sorting/remodeling of distinct molecular species en route to the plasma membrane.

Nano-electrospray ionization tandem mass spectrometry (nano-ESI-MS/MS) was employed to determine qualitative differences in the lipid molecular species composition of a comprehensive set of organellar membranes, isolated from a single culture of Saccharomyces cerevisiae cells. Remarkable differences in the acyl chain composition of biosynthetically related phospholipid classes were observed. Acyl chain saturation was lowest in phosphatidylcholine (15.4%) and phosphatidylethanolamine (PE; 16.2%), followed by phosphatidylserine (PS; 29.4%), and highest in phosphatidylinositol (53.1%). The lipid molecular species profiles of the various membranes were generally similar, with a deviation from a calculated average profile of approximately +/- 20%. Nevertheless, clear distinctions between the molecular species profiles of different membranes were observed, suggesting that lipid sorting mechanisms are operating at the level of individual molecular species to maintain the specific lipid composition of a given membrane. Most notably, the plasma membrane is enriched in saturated species of PS and PE. The nature of the sorting mechanism that determines the lipid composition of the plasma membrane was investigated further. The accumulation of monounsaturated species of PS at the expense of diunsaturated species in the plasma membrane of wild-type cells was reversed in elo3Delta mutant cells, which synthesize C24 fatty acid-substituted sphingolipids instead of the normal C26 fatty acid-substituted species. This observation suggests that acyl chain-based sorting and/or remodeling mechanisms are operating to maintain the specific lipid molecular species composition of the yeast plasma membrane.     

Amphiphilic amine-N-oxides with aliphatic alkyl chain act as efficient superoxide dismutase mimics, antioxidants and lipid peroxidation blockers in yeast

Amphiphilic 3-(alkanoylamino)propyldimethylamine-N-oxides with different length of the alkyl chain, i.e. different hydrophilic-lipophilic balance, act in micromolar concentrations as SOD mimics by lifting the inhibition of aerobic growth caused by SOD deletions in Saccharomyces cerevisiae. They also enhance the survival of sod mutants of S. cerevisiae exposed to the hydrophilic superoxide-generating prooxidant paraquat and the amphiphilic hydroperoxide-producing tert-butylhydroperoxide (TBHP), and largely prevent TBHP-induced peroxidation of isolated yeast plasma membrane lipids. Unlike the SOD-mimicking effect, the magnitude of these effects depends on the alkyl chain length of the amine-N-oxides, which incorporate into S. cerevisiae membranes, causing fluidity changes in both the hydrophilic surface part of the membrane and the membrane lipid matrix. Unlike wild-type strains, the membranes of sod mutants were found to contain polyunsaturated fatty acids; the sensitivity of the mutants to lipophilic pro-oxidants was found to increase with increasing content of these acids. sod mutants are useful in assessing pro- and antioxidant properties of different compounds.     

Lipid-dependent surface transport of the proton pumping ATPase: a model to study plasma membrane biogenesis in yeast

The proton pumping H+-ATPase, Pma1, is one of the most abundant integral membrane proteins of the yeast plasma membrane. Pma1 activity controls the intracellular pH and maintains the electrochemical gradient across the plasma membrane, two essential cellular functions. The maintenance of the proton gradient, on the other hand, also requires a specialized lipid composition of this membrane. The plasma membrane of eukaryotic cells is typically rich in sphingolipids and sterols. These two lipids condense to form less fluid membrane microdomains or lipid rafts. The yeast sphingolipid is peculiar in that it invariably contains a saturated very long-chain fatty acid with 26 carbon atoms. During cell growth and plasma membrane expansion, both C26-containing sphingolipids and Pma1 are first synthesized in the endoplasmatic reticulum from where they are transported by the secretory pathway to the cell surface. Remarkably, shortening the C26 fatty acid to a C22 fatty acid by mutations in the fatty acid elongation complex impairs raft association of newly synthesized Pma1 and induces rapid degradation of the ATPase by rerouting the enzyme from the plasma membrane to the vacuole, the fungal equivalent of the lysosome. Here, we review the role of lipids in mediating raft association and stable surface transport of the newly synthesized ATPase, and discuss a model, in which the newly synthesized ATPase assembles into a membrane environment that is enriched in C26-containing lipids already in the endoplasmatic reticulum. The resulting protein-lipid complex is then transported and sorted as an entity to the plasma membrane. Failure to successfully assemble this lipid-protein complex results in mistargeting of the protein to the vacuole.     

Localization of the membrane-associated thiol oxidase of rat kidney to the basal-lateral plasma membrane.

The localization of the membrane-associated thiol oxidase in rat kidney was investigated. Fractionation of the kidney cortex by differential centrifugation demonstrated that the enzyme is found in the plasma membrane. The crude plasma membrane was fractionated by density-gradient centrifugation on Percoll to obtain purified brush-border and basal-lateral membranes. Gamma-Glutamyltransferase, alkaline phosphatase and aminopeptidase M were assayed as brush-border marker enzymes, and (Na+ + K+)-stimulated ATPase was assayed as a basal-lateral-membrane marker enzyme. Thiol oxidase activity and distribution were determined and compared with those of the marker enzymes. Its specific activity was enriched 18-fold in the basal-lateral membrane fraction relative to its activity in the cortical homogenate, and its distribution paralleled that of (Na+ + K+)-stimulated ATPase. This association indicates that thiol oxidase is localized in the same fraction as (Na+ + K+)-stimulated ATPase, i.e. the basal-lateral region of the plasma membrane of the kidney tubular epithelium.     

Lipid composition and fluidity of plasma membranes isolated from corn (Zea mays L.) roots

Although the results of lipid analyses from several plant species have been available for many years a complete characterization of the corn root plasma membrane is still lacking. The present study provides a detailed analysis of individual lipids and a characterization of the membrane fluidity of corn (Zea mays L.) root plasma membranes isolated by phase-partitioning. Phospholipids (43.9 mol%), sterols (40.8 mol%), and sphingolipids in the form of glucocerebroside (6.8 mol%) constitute the major lipid classes. Stigmasterol (19.8 mol%), campesterol (13.0 mol%), phosphatidylcholine 15.8 mol%), and phosphatidylethanolamine (14.2 mol%) represent the most ubiquitous individual lipids. Hydroxy fatty acids make up 80.9 mol% and very long chain fatty acids are almost 78% of fatty acids in glucocerebroside. Hydroxy arachidic acid (20:0 h) and hydroxy lignoceric acid (24:0 h) are most prominent and glucocerebroside from corn root plasma membranes contains virtually no unsaturated fatty acids. Among the phospholipids only phosphatidylserine displayed a high proportion of very long chain fatty acids (e.g., behenic and lignoceric acid). Membrane fluidity was estimated by fluorescence anisotropy. Due to the high sterol content the plasma membrane of corn roots is relatively rigid.     

Thermotropic behavior and lateral distribution of very long chain sphingolipids

Sphingolipids containing very long acyl chains are abundant in certain specialized tissues and minor components of plasma membranes in most mammalian cells. There are cellular processes in which these sphingolipids are required, and the function seems to be mediated through sphingolipid-rich membrane domains. This study was conducted to explore how very long acyl chains of sphingolipids influence their lateral distribution in membranes. Differential scanning calorimetry showed that 24:0- and 24:1-sphingomyelins, galactosylceramides and glucosylceramides exhibited complex thermotropic behavior and partial miscibility with palmitoyl sphingomyelin. The T_m was decreased by about 20 °C for all 24:1-sphingolipids compared to the corresponding 24:0-sphingolipids. The ability to pack tightly with ordered and extended acyl chains is a necessity for membrane lipids to partition into ordered domains in membranes and thus the 24:1-sphingolipids appeared less likely to do so. Fluorescence quenching measurements showed that the 24:0-sphingolipids formed ordered domains in multicomponent membranes, both as the only sphingolipid and mixed with palmitoyl sphingomyelin. These domains had a high packing density which appeared to hinder the partitioning of sterols into them, as reported by the fluorescent cholesterol analog cholestatrienol. 24:0-SM was, however, better able to accommodate sterol than the glycosphingolipids. The 24:1-sphingolipids could, depending on head group structure, either stabilize or disrupt ordered sphingolipid/cholesterol domains. We conclude that very long chain sphingolipids, when present in biological membranes, may affect the physical properties of or the distribution of sterols between lateral domains. It was also evident that not only the very long acyl chain but also the specific molecular structure of the sphingolipids was of importance for their membrane properties.     

Alterations in the activities of hepatic plasma-membrane and microsomal enzymes during liver regeneration.

A marked increase in the activities of rat liver plasma-membrane (Na+ + K+)-stimulated ATPase and microsomal Ca2+-stimulated ATPase was observed 18h after partial hepatectomy. Lipid analyses for both membrane preparations reveal that in partially hepatectomized rats the cholesterol and sphingomyelin content are decreased with a subsequent decrease in the cholesterol/phospholipid molar ratio compared with those of sham-operated animals. Changes in the allosteric properties of plasma-membrane (Na+ + K+)-stimulated ATPase by F- (as reflected by changes in the Hill coefficient) indicated a fluidization of the lipid bilayer of both membrane preparations in 18 h-regenerating liver. The amphipathic dodecyl glucoside incorporated into the hepatic plasma membranes evoked a marked increase in the (Na+ + K+)-stimulated ATPase and 5'-nucleotidase activities. The lack of effect of the glucoside on the Lubrol-PX-solubilized 5'-nucleotidase indicates that changes in the activities of the membrane-bound enzymes caused by the glucoside are due to modulation of the membrane fluidity. Dodecyl glucoside appears to increase the membrane fluidity, evaluated through changes in the Hill coefficient for plasma-membrane (Na+ + K+)-stimulated ATPase. The biological significance of these data is discussed in terms of the differences and changes in the interaction of membrane-bound enzymes with membrane lipids during liver regeneration.     

Norepinephrine stimulation of lymphocyte ATPase by an alpha adrenergic receptor mechanism.

The effects of norepinephrine in interaction with adrenergic blocking compounds were studied on membrane adenosine triphosphatase (ATPase) activities of human lymphocytes and lymphoblasts. Sodium-potassium ion exchange pump activity was assayed by 86-Rb uptake and ATPase activity of membrane fractions was assayed by ADP and inorganic phosphate generation. The results of these studies indicate that norepinephrine acts by an alpha adrenergic mechanism to enhance membrane sodium-potassium ion exchange pump activity and ATPase activity. The pharmacologic and ionic dissection of the adrenergic sensitivity of ATPase activity indicates that this alpha adrenergic mechanism is related to membrane ATPase activities in addition to that associated with the ion exchange pump. Analysis of fractions obtained by sucrose gradients indicates that the action of norepinephrine is localized in the plasma membrane. Beta adrenergic stimulation was observed to inhibit ATPase activity. The complexity of adrenergic effects on membrane ATPase suggests interactions of hormone modulation of membrane nucleotide cyclases and transport-related ATPase enzymes.     

Ultrastructural localizations of adenosine triphosphatase activity in resting mammary gland.

Adenosine triphosphatase (ATPase) activity was localized at an ultrastructural level in the resting mammary glands of female BALB/c mice. A Mg++ dependent ATPase was localized in the plasma membranes of both the epithelial and myoepithelial cells of the mammary tubules. A second type of ATPase activity that was not Mg++-dependent but that was Na+ and K+ dependent was localized primarily in the plasma membranes of the myoepithelial cells. Preincubation with either ouabain or N-ethylmaleimide decreased the quantity of reaction product, indicating that both types of ATPase activity were sensitive to these inhibitors. Control media, containing adenosine triphosphate and Pb(NO3)2 without cations, demonstrated that the amount of nonezymatic hydrolysis was negligible. These differences in the cationic requirements for plasma membrane ATPase activity can be used to distinguish histochemically the epithelial from myoepithelial cells in mammary tissue.     

Characterization of nuclear membranes and endoplasmic reticulum isolated from plant tissue

Nuclei, nuclear membranes and rough endoplasmic reticulum (rER) were isolated from onion root tips and stems. Structural preservation and purity of the fractions was determined by electron microscopic and biochemical methods. Gross compositional data (protein, phospholipid, nonpolar lipids, sterols, RNA, DNA), phospholipid and fatty acid patterns, enzyme activities (ATPases, ADPase, IDPase, glucose-6-phosphatase, 5'-nucleotidase, acid phosphatase, and NADH- and NADPH-cytochrome C reductases), and cytochrome contents were determined. A stable, high salt-resistant attachment of some DNA with the nuclear membrane was observed as well as the association of some RNA with high salt-treated nuclear and rER membranes. The phospholipid pattern was identical for both nuclear and rER membranes and showed a predominance of lecithin (about 60%) and phosphatidyl ethanolamine (20-24%). Special care was necessary to minimize lipid degradation by phospholipases during isolations. Nonpolar lipids, mostly sterols and triglycerides, accounted for 35-45% of the membrane lipids. Sterol contents were relatively high in both membrane fractions (molar ratios of sterols to phospholipids ranged from 0.12 to 0.43). Sitosterol accounted for about 80% of the total sterols. Palmitic, oleic, and linoleic acids were the most prevalent acids in membrane-bound lipids as well as in storage lipids and occurred in similar proportions in phospholipids, triglycerides and free fatty acids of the membrane. About 80% of the fatty acids in membrane phospholipids and triglycerides were unsaturated. A cytochrome of the b5 type was characterized in these membranes, but P-450-like cytochromes could not be detected. Both NADH and NADPH-cytochrome c reductases were found in nuclear and rER membranes and appeared to be enriched in rER membranes. Among the phosphatases, Mg2+-ATPase and, to lesser extents, ADPase, IDPase and acid phosphatase activities occurred in the fractions, but significant amounts of monovalent ion-stimulated ATPase, 5'-nucleotidase and glucose-6-phosphatase activities did not. The results obtained emphasize that the close biochemical similarities noted between rER and nuclear membranes of animal cells extend to these fractions from plant cells.