Steady state kinetic studies of purified yeast plasma membrane proton-translocating ATPase

The plasma membrane H+-ATPase from bakers' yeast was purified and reconstituted with phosphatidylserine. The steady state kinetics of ATP hydrolysis catalyzed by the H+-ATPase were studied over a wide range of Mg2+ and ATP concentrations. Whereas MgATP was the substrate hydrolyzed, excess concentrations of either Mg2+ or ATP were inhibitory. The dependence of the steady state initial velocity of ATP hydrolysis on the concentration of MgATP at a fixed concentration of Mg2+ was sigmoidal rather than hyperbolic. This precluded mechanisms involving only activation and inhibition by Mg2+ and competitive inhibition by ATP. Two alternative interpretations of these results are: 1) the enzyme possesses multiple catalytic sites which interact cooperatively; or 2) the enzyme can exist in multiple conformational states which catalyze MgATP hydrolysis by parallel pathways. The rate laws for both mechanisms are identical so that the two mechanisms cannot be distinguished on the basis of the kinetic data. The data are well fit by the rate law for these mechanisms with the inclusion of competitive inhibition by Mg2+ and ATP and an independent inhibition site for Mg2+.     

NBD-labeled phosphatidylcholine and phosphatidylethanolamine are internalized by transbilayer transport across the yeast plasma membrane

The internalization and distribution of fluorescent analogs of phosphatidylcholine (M-C6-NBD-PC) and phosphatidylethanolamine (M-C6-NBD-PE) were studied in Saccharomyces cerevisiae. At normal growth temperatures, M-C6-NBD-PC was internalized predominantly to the vacuole and degraded. M-C6-NBD-PE was internalized to the nuclear envelope/ER and mitochondria, was not transported to the vacuole, and was not degraded. At 2 degrees C, both were internalized to the nuclear envelope/ER and mitochondria by an energy-dependent, N-ethylmaleimide-sensitive process, and transport of M-C6-NBD-PC to and degradation in the vacuole was blocked. Internalization of neither phospholipid was reduced in the endocytosis-defective mutant, end4-1. However, following pre-incubation at 37 degrees C, internalization of both phospholipids was inhibited at 2 degrees C and 37 degrees C in sec mutants defective in vesicular traffic. The sec18/NSF mutation was unique among the sec mutations in further blocking M-C6-NBD-PC translocation to the vacuole suggesting a dependence on membrane fusion. Based on these and previous observations, we propose that M-C6-NBD-PC and M-C6-NBD-PE are transported across the plasma membrane to the cytosolic leaflet by a protein-mediated, energy-dependent mechanism. From the cytosolic leaflet, both phospholipids are spontaneously distributed to the nuclear envelope/ER and mitochondria. Subsequently, M-C6-NBD-PC, but not M-C6-NBD-PE, is sorted by vesicular transport to the vacuole where it is degraded by lumenal hydrolases.     

Regulation of transbilayer plasma membrane phospholipid asymmetry

Lipids in biological membranes are asymmetrically distributed across the bilayer; the amine-containing phospholipids are enriched on the cytoplasmic surface of the plasma membrane, while the choline-containing and sphingolipids are enriched on the outer surface. The maintenance of transbilayer lipid asymmetry is essential for normal membrane function, and disruption of this asymmetry is associated with cell activation or pathologic conditions. Lipid asymmetry is generated primarily by selective synthesis of lipids on one side of the membrane. Because passive lipid transbilayer diffusion is slow, a number of proteins have evolved to either dissipate or maintain this lipid gradient. These proteins fall into three classes: 1) cytofacially-directed, ATP-dependent transporters ("flippases"); 2) exofacially-directed, ATP-dependent transporters ("floppases"); and 3) bidirectional, ATP-independent transporters ("scramblases"). The flippase is highly selective for phosphatidylserine and functions to keep this lipid sequestered from the cell surface. Floppase activity has been associated with the ABC class of transmembrane transporters. Although they are primarily nonspecific, at least two members of this class display selectivity for their substrate lipid. Scramblases are inherently nonspecific and function to randomize the distribution of newly synthesized lipids in the endoplasmic reticulum or plasma membrane lipids in activated cells. It is the combined action of these proteins and the physical properties of the membrane bilayer that generate and maintain transbilayer lipid asymmetry.     

Mathematical modelling of lipid transbilayer movement in the human erythrocyte plasma membrane

A model is presented to simulate transverse lipid movement in the human erythrocyte membrane. The model is based on a system of differential equations describing the time-dependence of phospholipid redistribution and the steady state distribution between the inner and outer membrane monolayer. It takes into account several mechanisms of translocation: (i) ATP-dependent transport via the aminophospholipid translocase; (ii) protein-mediated facilitated and (iii) carrier independent transbilayer diffusion. A reasonable modelling of the known lipid asymmetry could only be achieved by introducing mechanism (iii). We have called this pathway the compensatory flux, which is proportional to the gradient of phospholipids between both membrane leaflets. Using realistic model parameters, the model allows the calculation of the transbilayer motion and distribution of endogenous phospholipids of the human erythrocyte membrane for several biologically relevant conditions. Moreover, the model can also be applied to experiments usually performed to assess phospholipid redistribution in biological membranes. Thus, it is possible to simulate transbilayer motion of exogenously added phospholipid analogues in erythrocyte membranes. Those experiments have been carried out here in parallel using spin labeled lipid analogues. The general application of this model to other membrane systems is outlined.Abbreviations PBS phosphate buffered saline - DFP diisopropyl fluorophosphate - ESR electron spin resonance - RBC red blood cells - PC phosphatidylcholine - PE phosphatidylethanolamine - PS phosphatidylserine - SM sphingomyelin - (0,2)PC 1-palmitoyl-2(4doxylpentanoyl)-PC - (0,2)PE 1-palmitoyl-2(4-doxylpentanoyl)-PE - (0,2) PS 1-palmitoyl-2(4-doxylpentanoyl)-PS     

The transbilayer distribution of phosphatidylethanolamine in erythroid plasma membranes during erythropoiesis

Fluorescamine was used to assess the transbilayer distribution of phosphatidylethanolamine in the plasma membrane of murine erythroid progenitor cells, CFU-E (colony-forming unit erythroid), at different stages of their differentiation pathway. Intact cells were exposed to increasing concentrations of fluorescamine and the amount of labeled phosphatidylethanolamine was determined by measuring the fluorescence intensity of its fluorescamine derivative. A semilogarithmic plot of the dose-response curve revealed three different pools of phosphatidylethanolamine, representing its fractions in, respectively, the inner- and outer monolayers of the plasma membrane and subcellular membrane systems. These results show that 9-11% of the total cellular phosphatidylethanolamine is present in the outer leaflet and 9-10% of it is located in the inner leaflet of the plasma membrane in early as well as late erythroblasts. This symmetric distribution of phosphatidylethanolamine over the two halves of the bilayer in the plasma membrane of CFU-E is very similar to that observed earlier in the plasma membrane of friend erythroleukaemic cells (Rawyler, Van der Schaft, Roelofsen and Op den Kamp (1985) Biochemistry 24, 1777-1783). These observations imply that the characteristic asymmetric distribution of phosphatidylethanolamine, as is found in mature erythrocytes, is accomplished at a very late stage of erythropoiesis and possibly during enucleation of the cells or shortly thereafter.     

Rapid transbilayer movement of fluorescent phospholipid analogues in the plasma membrane of endocytosis-deficient yeast cells does not require the Drs2 protein.

Evidence is presented that endocytosis-deficient Saccharomyces cerevisiae end4 yeast cells rapidly internalize the fluorescent phospholipid analogues 1-palmitoyl-2-{6-[7-nitro-2,1, 3-benzoxadiazol-4-yl(NBD)amino] caproyl}phosphatidylcholine (P-C6-NBD-PtdCho) and P-C6-NBD-phosphatidylserine (P-C6-NBD-PtdSer). Both analogues redistributed between the exoplasmic and cytoplasmic leaflet with a half-time of < 15 min at 0 degrees C. The plateau of internalized analogues was about 70%. Transbilayer movement is probably protein-mediated, as the flip-flop of both analogues was very slow in liposomes composed of plasma-membrane lipids. Rapid analogue internalization was not abolished on depletion of intracellular ATP by about 90%. For P-C6-NBD-PtdCho only was a moderate decrease in the plateau of internalized analogues of about 20% observed, while that of P-C6-NBD-PtdSer was not affected. The Drs2 protein plays only a minor role, if any, in the rapid transbilayer movement of analogues in S. cerevisiae end4 cells. In S. cerevisiae end4 Deltadrs2 cells harbouring both an end4 allele and a drs2 null allele, about 60% and 50% of P-C6-NBD-PtdCho and P-C6-NBD-PtdSer, respectively, became internalized within 15 min at 0 degrees C. The preferential orientation of P-C6-NBD-PtdSer to the cytoplasmic leaflet is in qualitative agreement with the sequestering of endogenous phosphatidylserine to the cytoplasmic leaflet, as assessed by binding of annexin V. Virtually no binding of annexin V to spheroplasts of the parent wild-type strain or the mutant strains was observed. Likewise, no difference in the exposure of endogenous aminophospholipids to the exoplasmic leaflet between these strains was found by labelling with trinitrobenzenesulfonic acid. Thus, lipid asymmetry, at least of aminophospholipids, was preserved in S. cerevisiae end4 cells independently of the presence of the Drs2 protein.     

Polyunsaturated fatty acids alter sterol transbilayer domains in LM fibroblast plasma membrane

Sterols are asymmetrically distributed between the leaflets of animal cell plasma membranes. Although transbilayer migration of sterols is extremely rapid, s to min, previous experimental manipulations have not altered their transmembrane steady-state distribution. However, the effect of polyunsaturated fatty acids has not been reported. When cultured in a lipid-free, chemically defined culture medium, LM fibroblasts do not synthesize polyunsaturated fatty acids but will incorporate polyunsaturated fatty acids into their plasma membranes if supplied in the medium. Sterol transbilayer distribution in LM plasma membranes was determined from quenching of fluorescence of dehydroergosterol by trinitrophenyl groups selectively attached to the exofacial leaflet. When cells are cultured in lipid-free media, 28.1% of the plasma membrane sterol is located in the exofacial (outside) leaflet. In contrast, when cells are cultured with linoleate- or linolenate-supplemented medium, 71.8% and 75.5% of the plasma membrane sterol is exofacial, respectively.     

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.     

Immunochemical analysis of the plasma membrane from baker's yeast Saccharomyces cerevisiae

Yeast plasma membranes have been isolated from homogenized yeast cells, identified as pure plasma membrane vesicles which were used as antigens. By crossed immunoelectrophoresis with anti-membrane immunoglobulins, 17 discrete antigens have been detected in Triton X-100 extracts from plasma membranes. Three different immunoabsorption experiments were performed with : a) isolated membranes exposing the cytoplasmic surfaces (PS) and the external surfaces (ES), b) yeast protoplasts exposing only antigenic determinants on the ES, c) lysed protoplasts which had been saturated on the ES with antibodies prior to lysis. These absorption experiments demonstrated that seven of the antigens are expressed on the ES while eight immunogens expose antigenic determinants on the PS. Four of the principal immunoprecipitates are not affected by absorption with surface antigens whereas two of the antigens indicate transmembrane characteristics. Of these 17 immunoprecipitates four were shown by zymograms to possess enzymatic activities: ATPase (EC 3.6.1.3) and NADH-dehydrogenase (EC 1.8.99.3) (three separate components). Three of these enzymes are expressed on the PS, and one NADH-dehydrogenase exposes determinants on the ES of the protoplasts. The presence of antigens on the PS of the plasma membrane could also be demonstrated on micrographs by the indirect ferritin-antibody labeling technique followed by freeze-etching and shadowing of the membranes.     

Role of polyunsaturated fatty acids and lipid peroxidation in LM fibroblast plasma membrane transbilayer structure

The effects of polyunsaturated fatty acids and lipid peroxidation on LM fibroblast plasma membrane individual leaflet sterol distribution and structural order were examined. The cytofacial (inner) leaflet was more rigid and contained more sterol than the exofacial (outer) leaflet. The static (limiting anisotropy) and dynamic (rotational relaxation time) structural components of diphenylhexatriene (DPH) motion in each leaflet were determined by phase and modulation fluorometry measurements combined with leaflet-specific quenching by trinitrophenyl groups. Polyunsaturated fatty acids, incorporated into the membrane phospholipids by culture medium supplementation, decreased the limiting anisotrophy of DPH in the cytofacial but not the exofacial leaflet thereby abolishing the transbilayer difference in fluidity. Peroxidation by Fe(II) + H2O2 resulted in a rigidification (increase in limiting anisotropy and rotational relaxation time) of the plasma membrane exofacial leaflet, regardless of whether the membranes contained saturated and monounsaturated fatty acids or were enriched in either linoleate or linolenate. The structure of the cytofacial leaflet reported by DPH was unaffected. Plasma membrane transbilayer sterol distribution, measured by leaflet-specific quenching of dehydroergosterol fluorescence, indicated that 20-28% of the sterol was localized in the exofacial leaflet. Polyunsaturated fatty acid supplementation of LM fibroblasts resulted in a complete reversal of plasma membrane transbilayer sterol distribution (72-76% exofacial leaflet). Sterol transbilayer distribution between the membrane leaflets was completely resistant to alteration by exposure to crosslinking agents and peroxidation in control plasma membranes and by peroxidation in linoleate- or linolenate-supplemented membranes.     

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.     

高效液相色谱法测定酵母中麦角固醇含量

本文建立了酵母中麦角固醇含量高效液相色谱测定方法。其色谱条件为,色谱柱HYPersilBDSC185u反相柱,流动相为甲醇:水(97∶3),紫外检测波长为283nm。酵母样加碱乙醇皂化、提取、洗涤、蒸干、定量测定。结果表明:标准曲线范围是002—08mg/ml线性良好,最低限量为001mg/ml;日内及日间RSD(n=4)分别在21～40%和24～48%,回收率为960～980%。本方法操作简便、准确、可靠。     

Steady state analysis of mitochondrial RNA after growth of yeast Saccharomyces cerevisiae under catabolite repression and derepression

The steady state levels of mitochondrial rRNAs, 5 tRNAs, the 9 S RNA, and the RNA products from the genes coding for subunits 6 and 9 of the ATP synthase, cytochrome b, and subunit 1 of cytochrome oxidase have been determined after growth of yeast under conditions of respiratory repression or derepression. The analysis indicates that the mitochondrial rRNAs are present in 2000 or 9000 copies/cell in repressed or derepressed yeast, respectively. The levels of the other RNAs also differed to a similar extent, with the exception of the level of the tRNAfMet which differs by only 1.7-fold. The levels of the individual protein coding RNAs varied from 480 copies/cell for the Oli-1 RNA to 100 copies/cell for the Oli-2 RNA under derepressive conditions and from 130 copies/cell to 33 copies/cell for the same RNAs in glucose repressive conditions. The levels of the tRNAs varied even more markedly, ranging from 4200 copies/cell for the tRNAPhe to 240 copies/cell for the tRNACys after growth in derepressive conditions and from 800 copies/cell for the tRNAfMet to 30 copies/cell for the tRNACys of glucose repressed yeast. These results indicate that glucose repression uniformly decreases the levels of the individual mitochondrial RNAs studied. This decrease is related to a lower synthesis of mitochondrial RNA in the glucose repressed cells as compared to derepressed cells.     

Topology of the yeast plasma membrane proton-translocating ATPase

Four proteases have been used to assess the topology of the H+-ATPase from Saccharomyces cerevisiae reconstituted into phosphatidylserine vesicles. Limited proteolysis by trypsin and alpha-chymotrypsin inactivates the enzyme and produces stable, membrane-bound fragments. Sequence analyses of these peptides have located the peptide bonds hydrolyzed. The labile bonds are on opposite sides of a central hydrophilic domain containing consensus sequences for the site of phosphorylation and fluorescein isothiocyanate binding of several related ATPases. Limited proteolysis of the ATPase by elastase cuts approximately 50 amino acids from the C terminus, leaving the remaining membrane-bound fragments active. Proteolysis by carboxypeptidase Y in the presence and absence of detergent suggests that the C terminus is on the inside of the vesicle in this reconstitution. A model for the transmembrane arrangement of the polypeptide is proposed. In this model, the C terminus is on the inside of the vesicle, the N terminus is on the outside, the ATP binding region is on the outside, and the polypeptide passes through the membrane a minimum of five times.     

The isolation of plasma membrane and characterisation of the plasma membrane ATPase from the yeast Candida albicans

Plasma membrane ghosts were isolated from Candida albicans ATCC 10261 yeast cells following stabilisation of spheroplasts with concanavalin A, osmotic lysis and Percoll density gradient centrifugation. Removal of extrinsic proteins with NaCl and methyl alpha-mannoside gave increased ATPase and chitin synthase specific activities in the resultant plasma membrane fraction. Sonication of this fraction yielded unilamellar plasma membrane vesicles which exhibited ATPase and chitin synthase specific activities of 4.5-fold and 3.0-fold, respectively, over those of the plasma membrane ghosts. ATPase activity in the membrane ghosts was optimal at pH 6.4, showed high substrate specificity (for Mg X ATP) and was inhibited 80% by sodium vanadate but less than 4% by oligomycin and azide. The effects of a range of other inhibitors were also characterised. Temperature effects of ATPase activity were marked, with a maximum at 35 degrees C. Breaks in the Arrhenius plot, at 12.2 degrees C and 28.9 degrees C, coincided with endothermic heat flow peaks detected by differential scanning calorimetry. ATPase was solubilised from the plasma membranes with Zwittergent in the presence of glycerol and phenylmethylsulphonyl fluoride and partially purified by glycerol density gradient centrifugation. The solubilised enzyme hydrolysed Mg X ATP at Vmax = 20 mumol X min-1 X mg-1 in the presence of phospholipids, with optimal activity at pH 6.0--6.5.     

Ethanol tolerance and the variation of plasma membrane composition of yeast floc populations with different size distribution

The ethanol tolerance of a self-flocculating yeast strain SPSC01 was investigated in an oxygen-limited fed-batch bioreactor. Employing Focused Beam Reflectance Measurement (FBRM) on-line monitoring system, four yeast floc populations with the average size ranging from 100 to 400mum were obtained. It was found that ethanol tolerance increased with the increasing floc size in the 100, 200, and 300mum floc populations, while increasing the average floc size further to 400mum resulted in lower ethanol tolerance. Examination of the membrane composition of different floc populations revealed that the plasma membrane composition of the floc populations was significantly different in the contents of ergosterol, phosphatidylinositol, as well as phospholipid palmitoleic acid. What's more, the plasma membrane of more ethanol tolerant floc population was less permeable when subjected to 15% (v/v) ethanol shock treatment, and the plasma membrane ATPase activities were higher in the floc populations with higher ethanol tolerance. These results indicate that the average size distribution of the floc populations exerted great influence on the physiological status of yeast cells during the ethanol production process, leading to the changes in plasma membrane composition that contributed to improved ethanol tolerance in self-flocculating yeast SPSC01.