Unbalanced phospholipid composition of <Emphasis Type="Italic">Escherichia coli</Emphasis> membranes affects P<Subscript>PHO</Subscript> promoter activity

The activity of phosphatase P_PHO or arabinose P_BAD promoters of Escherichia coli has been studied as dependent on the content of zwitterionic phosphatidylethanolamine (PE) and anionic phospholipids in membranes. In the absence of PE or upon a significant decrease in the content of anionic phospholipids, the activity of the P_PHO promoter (but not that of the P_BAD promoter) was inhibited. Since the P_PHO promoter belongs to the Pho regulon, a member of the family of two-component regulatory systems of signal transduction that have membrane sensors, the regulation of gene expression by phospholipids is presumed to be accomplished through a membrane sensor.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 2, 2005, pp. 294–302.Original Russian Text Copyright © 2005 by Golovastov, Krasovskaya, Anissimova, Shchukin, Badyakina, Nesmeyanova.Deceased.     

Membrane properties induced by anionic phospholipids and phosphatidylethanolamine are critical for the membrane binding and catalytic activity of human cytochrome P450 3A4

Human cytochrome P450 (CYP) 3A4, a membrane anchoring protein, is the major CYP enzyme present in both liver and small intestine. The enzyme plays a major role in the metabolism of many drugs and procarcinogens. The roles of individual phospholipids and membrane properties in the catalytic activity, membrane binding, and insertion into the membrane of CYP3A4 are poorly understood. Here we report that the catalytic activity of testosterone 6beta-hydroxylation, membrane binding, and membrane insertion of CYP3A4 increase as a function of anionic phospholipid concentration in the order phosphatidic acid (PA) > phosphatidylserine (PS) in a binary system of phosphatidylcholine (PC)/anionic phospholipid and as a function of phosphatidylethanolamine (PE) content in ternary systems of PC/PE/PA or PC/PE/PS having a fixed concentration of anionic phospholipids. These results suggest that PA and PE might help the binding of CYP3A4 to the membrane and the interaction with NPR. Cytochrome b(5) (b(5)) and apolipoprotein b(5) further enhanced the testosterone 6beta-hydroxylation activities of CYP3A4 in all tested phospholipids vesicles with various compositions. Phospholipid-dependent changes of the CYP3A4 conformation were also revealed by altered Trp fluorescence and CD spectra. We also found that PE induced the formation of anionic phospholipid-enriched domains in ternary systems using extrinsic fluorescent probes incorporated into lipid bilayers. Taken together, it can be suggested that the chemical and physical properties of membranes induced by anionic phospholipids and PE are critical for the membrane binding and catalytic activity of CYP3A4.     

Phospholipid-assisted protein folding: phosphatidylethanolamine is required at a late step of the conformational maturation of the polytopic membrane protein lactose permease.

Previously we presented evidence that phosphatidylethanolamine (PE) acts as a molecular chaperone in the folding of the polytopic membrane protein lactose permease (LacY) of Escherichia coli. Here we provide more definitive evidence supporting the chaperone properties of PE. Membrane insertion of LacY prevents its irreversible aggregation, and PE participates in a late step of conformational maturation. The temporal requirement for PE was demonstrated in vitro using a coupled translation-membrane insertion assay that allowed the separation of membrane insertion from phospholipid-assisted folding. LacY was folded properly, as assessed by recognition with conformation-specific monoclonal antibodies, when synthesized in the presence of PE-containing inside-out membrane vesicles (IOVs) or in the presence of IOVs initially lacking PE but supplemented with PE synthesized in vitro either co- or post-translationally. The presence of IOVs lacking PE and containing anionic phospholipids or no addition of IOVs resulted in misfolded or aggregated LacY, respectively. Therefore, critical folding steps occur after membrane insertion dependent on the interaction of LacY with PE to prevent illicit interactions which lead to misfolding of LacY.     

Coincident exposure of phosphatidylethanolamine and anionic phospholipids on the surface of irradiated cells

The major anionic phospholipid, phosphatidylserine (PS), and the neutral phospholipid, phosphatidylethanolamine (PE), are largely confined to the inner leaflet of the plasma membrane bilayer in mammalian cells under normal conditions. This asymmetry is lost when cells undergo apoptosis, become activated, or are exposed to irradiation, reactive oxygen species or certain drugs. It is not known whether exposure of anionic phospholipids (APLs) and PE occurs simultaneously or in the same region of the plasma membrane. Here we examined the coincidence of exposure of APLs and PE on the surface of bovine aortic endothelial cells and NS0 myeloma cells after irradiation. The cells were irradiated (5 Gy) and stained for APLs and PE using liposomes coated with either an Fab′ fragment of a PS-binding antibody (bavituximab) or a PE-binding peptide (duramycin). Using live cell imaging and flow cytometry, we showed that irradiation leads to synchronous externalization of APLs and PE. The time course of appearance of APLs and PE on the cell surface was the same and the two phospholipid types remained colocalized over time. Distinct patches double positive for APLs and PE were visible. Larger areas of APLs and PE appeared to have detached from the cytoskeleton to form membrane blebs which protruded and drifted on the cell surface. We conclude that APLs and PE coincidently appear on the external leaflet of the plasma membrane of cells after irradiation. Probably, this is because PE and the major APL, PS, share common regulatory mechanisms of translocation.     

水分逆境对吊兰叶片脂质组成的影响

测定了吊兰（ Ｃｈｌｏｒｏｐｈｙｔｕｍ ｃｏｍｏｓｕｍ） 在干旱、正常浇水和渍水三种供水条件下叶片的磷脂组成、膜脂和总磷脂的脂肪酸组成,以及磷脂中４ 种主要组分ＰＧ、ＰＥ、ＰＣ 和ＰＩ的脂肪酸组成,观察到干旱使磷脂中ＰＥ 的相对含量增加,ＰＥ 脂肪酸中１６ ：０ 明显减少,而膜脂、总磷脂和ＰＣ、ＰＩ中饱和脂肪酸增加,但ＰＧ脂肪酸组成变化很小     

水分逆境对吊兰叶片膜质组成的影响

测定了吊兰(Chlorophytum comosum)在干旱、正常浇水和渍水三种供水条件下叶片的磷脂组成、膜脂和总磷脂的脂肪酸组成,以及磷脂中4种主要组分PG、PE、PC和PI的脂肪酸组成,观察到干旱使磷脂中PE的相对含量增加,PE脂肪酸中16:0明显减少,而膜脂,总磷脂和PC、PI中饱和脂肪酸增加,但PG脂肪酸组成变化很小。     

Role of anionic lipid in bacterial membranes

The major phospholipids of Bacillus stearothermophilus are phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and cardiolipin (CL). Under the growth conditions used in this study the concentration of anionic lipid (PG + CL) was determined by the pH of the culture medium. Cells grown in a complex medium at pH 5.8, 7.0, and 8.0 contained 17, 29 and 36 nmol of anionic (PG + CL) lipid/mg cell (dry weight). The concentration of the zwitterionic lipid phosphatidylethanolamine (PE) was 17-20 nmol/mg cell (dry weight) under all conditions. Analysis of isolated membrane preparations suggested that the amount of anionic lipid per unit area of membrane increased as the pH of the growth medium was increased. Membranes from cells grown at pH 5.8 and 8.0 contained 130 and 320 nmol anionic lipid/mg membrane protein, respectively. Phosphatidylethanolamine appeared to be localized on the inner membrane surface in cells grown under all conditions. Increasing the ionic strength of the culture medium by the addition of NaCl or KCl had little effect on growth at pH 5.8 but inhibited growth at pH 7 and 8. It was concluded that anionic phospholipid plays an important physiological role in maintaining an acidic pH at the outer membrane surface.     

Anionic phospholipids stimulate an arachidonoyl-hydrolyzing phospholipase A2 from macrophages and reduce the calcium requirement for activity

Mechanisms involved in regulating the activity of intracellular phospholipase A2 enzymes that function in eicosanoid and platelet-activating factor production are poorly understood. The properties of the substrate in the membrane may play a role in modulating phospholipase A2 activity. In this study, the effect of anionic phospholipids, diacylglycerol (DAG) and phosphatidylethanolamine (PE) on the activity of a partially purified, intracellular, arachidonoyl-hydrolyzing phospholipase A2 from the macrophage cell line, RAW 264.7 was studied. For these experiments phospholipase A2 activity was assayed in the presence of 1 microM calcium by measuring the hydrolysis of [3H]arachidonic acid from sonicated dispersions of the ether-linked substrate, 1-O-hexadecyl-2[3H]arachidonoylglycerophosphocholine. All the anionic phospholipids tested, including phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylinositol (PI) and phosphatidylinositol-4,5-bisphosphate (PIP2), stimulated phospholipase A2 activity. At the lowest concentration of anionic phospholipids tested. PIP2 was the most stimulatory, resulting in a 7-fold increase in phospholipase A2 activity at 1 mol%. Co-dispersion of either DAG or PE with the substrate also induced a dose-dependent increase in phospholipase A2 activity, whereas sphingomyelin was inhibitory suggesting that the phospholipase A2 more readily hydrolyzed the ether-linked substrate when there was a decrease in the packing density of the bilayer. PIP2, together with either DAG or PE, synergistically stimulated phospholipase A2 activity by about 20-fold, and dramatically decreased the calcium concentration (from mM to nM) required for full activity of the enzyme. The results of this study demonstrate that the presence of anionic phospholipids and the packing characteristics of the bilayer can have pronounced effects on the activity and calcium requirement of an intracellular, arachidonoyl-hydrolyzing phospholipase A2 from macrophages.     

Interaction of the K+ channel KcsA with membrane phospholipids as studied by ESI mass spectrometry

In this study we have used electrospray ionization mass spectrometry (ESI-MS) to investigate interactions between the bacterial K(+) channel KcsA and membrane phospholipids. KcsA was reconstituted into lipid vesicles of variable lipid composition. These vesicles were directly analyzed by ESI-MS or mixed with trifluoroethanol (TFE) before analysis. In the resulting mass spectra, non-covalent complexes of KcsA and phospholipids were observed with an interesting lipid specificity. The anionic phosphatidylglycerol (PG), and, to a lesser extent, the zwitterionic phosphatidylethanolamine (PE), which both are abundant bacterial lipids, were found to preferentially associate with KcsA as compared to the zwitterionic phosphatidylcholine (PC). These preferred interactions may reflect the differences in affinity of these phospholipids for KcsA in the membrane.     

Anionic phospholipids are determinants of membrane protein topology.

The orientation of many membrane proteins is determined by the asymmetric distribution of positively charged amino acid residues in cytoplasmic and translocated loops. The positive-inside rule states that loops with large amounts of these residues tend to have cytoplasmic locations. Orientations of constructs derived from the inner membrane protein leader peptidase from Escherichia coli were found to depend on the anionic phospholipid content of the membrane. Lowering the contents of anionic phospholipids facilitated membrane passage of positively charged loops. On the other hand, elevated contents of acidic phospholipids in the membrane rendered translocation more sensitive to positively charged residues. The results demonstrate that anionic lipids are determinants of membrane protein topology and suggest that interactions between negatively charged phospholipids and positively charged amino acid residues contribute to the orientation of membrane proteins.     

Phosphatidylethanolamine Is a Key Regulator of Membrane Fluidity in Eukaryotic Cells

Adequate membrane fluidity is required for a variety of key cellular processes and in particular for proper function of membrane proteins. In most eukaryotic cells, membrane fluidity is known to be regulated by fatty acid desaturation and cholesterol, although some cells, such as insect cells, are almost devoid of sterol synthesis. We show here that insect and mammalian cells present similar microviscosity at their respective physiological temperature. To investigate how both sterols and phospholipids control fluidity homeostasis, we quantified the lipidic composition of insect SF9 and mammalian HEK 293T cells under normal or sterol-modified condition. As expected, insect cells show minimal sterols compared with mammalian cells. A major difference is also observed in phospholipid content as the ratio of phosphatidylethanolamine (PE) to phosphatidylcholine (PC) is inverted (4 times higher in SF9 cells). In vitro studies in liposomes confirm that both cholesterol and PE can increase rigidity of the bilayer, suggesting that both can be used by cells to maintain membrane fluidity. We then show that exogenously increasing the cholesterol amount in SF9 membranes leads to a significant decrease in PE:PC ratio whereas decreasing cholesterol in HEK 293T cells using statin treatment leads to an increase in the PE:PC ratio. In all cases, the membrane fluidity is maintained, indicating that both cell types combine regulation by sterols and phospholipids to control proper membrane fluidity.     

Dependence of prePhoA-phospholipid interaction in vivo and in vitro on charge of signal peptide N-terminus and content of anionic phospholipids in membranes

Replacement of the positively charged signal peptide with neutral or negatively charged peptides due to substitution of Lys(–20) in the N-terminal region of the signal peptide leads to decreases in the rate of prePhoA membrane translocation in vivo and in the efficiency of prePhoA insertion into liposomes in vitro. The effect of anionic phospholipids on prePhoA insertion into model membranes is determined by the signal peptide N-terminus charge, while the dependence of prePhoA translocation across the cytoplasmic membrane in vivo is not, under the studied variations in the content of anionic phospholipids. This is evidence of the possibility of direct electrostatic interaction between the signal peptide N-terminus and anionic phospholipids, which in vivo, however, seems to involve some proteins of the Sec machinery.     

Changes in the composition of anionic membrane phospholipids influence the protein secretion and biogenesis of cell envelope in Escherichia coli

The secretion of alkaline phosphatase (PhoA) and peculiarities of biogenesis of the cell envelope were studied in Escherichia coli strains HD30/pHD 102 and HDL11 with controlled synthesis of anionic phospholipids, phosphatidylglycerol, and cardiolipin. Inactivation of the pgsA gene encoding the synthesis of anionic phospholipids or changes in the regulation of its expression by an environmental factor caused changes in the metabolism and composition of membrane phospholipids, which resulted in a decrease in the secretion of alkaline phosphatase through the cytoplasmic membrane and an increase in PhoA secretion from the periplasm into the culture medium. A conforming increase was observed in exopolysaccharide secretion, as well as a decrease in the contents of lipopolysaccharide and lipopolyprotein of the outer membrane that determine the membrane barrier properties. The results obtained testify that anionic phospholipids play a significant role in protein secretion and are probably involved in the interrelation between the protein secretion and biogenesis of cell envelope components.     

Anionic lipids enriched at the ExPortal of Streptococcus pyogenes

The ExPortal of Streptococcus pyogenes is a membrane microdomain dedicated to the secretion and folding of proteins. We investigated the lipid composition of the ExPortal by examining the distribution of anionic membrane phospholipids. Staining with 10-N-nonyl-acridine orange revealed a single microdomain enriched with an anionic phospholipid whose staining characteristics and behavior in a cardiolipin-deficient mutant were characteristic of phosphatidylglycerol. Furthermore, the location of the microdomain corresponded to the site of active protein secretion at the ExPortal. These results indicate that the ExPortal is an asymmetric lipid microdomain, whose enriched content of anionic phospholipids may play an important role in ExPortal organization and protein trafficking.     

Effect of Membrane Phospholipid Composition and Charge of the Signal Peptide of Escherichia coli Alkaline Phosphatase on Efficiency of Its Secretion

The secretion of the Escherichia coli alkaline phosphatase with a different charge of signal peptide due to replacement of positively charged Lys(–20) has been studied depending on the phospholipid composition of the membranes and the activity of the translocational ATPase—protein SecA. Changing the signal peptide charge, along with a change in phospholipid composition, has been shown to reduce the efficiency of secretion. In the absence of phosphatidylethanolamine the membrane contains anionic phospholipids only, and the dependence of secretion on the signal peptide charge decreases. The dependence of secretion on membrane phospholipid composition and the signal peptide charge is also determined by the activity of SecA protein. If SecA is inactivated by sodium azide, then the dependence of secretion on anionic phospholipids increases; on the contrary, higher content of anionic phospholipids (in the absence of phosphatidylethanolamine) decreases the dependence of secretion on the SecA activity. The results suggest a direct interaction of positively charged signal peptide with negatively charged membrane phospholipids under initiation of secretion and also interdependent contribution of the signal peptide charge, anionic phospholipids, and translocational ATPase to secretion.     

Effects of phospholipid composition on MinD-membrane interactions in vitro and in vivo

The peripheral membrane ATPase MinD is a component of the Min system responsible for correct placement of the division site in Escherichia coli cells. By rapidly migrating from one cell pole to the other, MinD helps to block unwanted septation events at the poles. MinD is an amphitropic protein that is localized to the membrane in its ATP-bound form. A C-terminal domain essential for membrane localization is predicted to be an amphipathic alpha-helix with hydrophobic residues interacting with lipid acyl chains and cationic residues on the opposite face of the helix interacting with the head groups of anionic phospholipids (Szeto, T. H., Rowland, S. L., Rothfield, L. I., and King, G. F. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 15693-15698). To investigate whether E. coli MinD displays a preference for anionic phospholipids, we first examined the localization dynamics of a green fluorescent protein-tagged derivative of MinD expressed in a mutant of E. coli that lacks phosphatidylethanolamine. In these cells, which contain only anionic phospholipids (phosphatidylglycerol and cardiolipin), green fluorescent protein-MinD assembled into dynamic focal clusters instead of the broad zones typical of cells with normal phospholipid content. In experiments with liposomes composed of only zwitterionic, only anionic, or a mixture of anionic and zwitterionic phospholipids, purified MinD bound to these liposomes in the presence of ATP with positive cooperativity with respect to the protein concentration and exhibited Hill coefficients of about 2. Oligomerization of MinD on the liposome surface also was detected by fluorescence resonance energy transfer between MinD molecules labeled with different fluorescent probes. The affinity of MinD-ATP for anionic liposomes as well as liposomes composed of both anionic and zwitterionic phospholipids increased 9- and 2-fold, respectively, relative to zwitterionic liposomes. The degree of acyl chain unsaturation contributed positively to binding strength. These results suggest that MinD has a preference for anionic phospholipids and that MinD oscillation behavior, and therefore cell division site selection, may be regulated by membrane phospholipid composition.     

Changes in the composition of anionic membrane phospholipids influence protein secretion and cell envelope biogenesis in Escherichia coli

The secretion of alkaline phosphatase (PhoA) and peculiarities of biogenesis of the cell envelope were studied in Escherichia coli strains HD30/pHD102 and HDL11 with controlled synthesis of the anionic phospholipids, phosphatidylglycerol and cardiolipin. Inactivation of the pgsA gene responsible for the synthesis of anionic phospholipids or changes in the regulation of its expression by an environmental factor caused changes in the metabolism and composition of membrane phospholipids, which resulted in a decrease in the secretion of alkaline phosphatase through the cytoplasmic membrane and an increase in PhoA secretion from the periplasm into the culture medium. An increase was observed in exopolysaccharide secretion, as well as a decrease in the contents of the outer membrane lipopolysaccharides and lipopolyproteins, which determine its barrier properties. The results obtained show that anionic phospholipids play a significant role in protein secretion and are probably involved in the interrelation between protein secretion and biogenesis of cell envelope components.__________Translated from Mikrobiologiya, Vol. 74, No. 2, 2005, pp. 179–184.Original Russian Text Copyright © 2005 by Anisimova, Badyakina, Vasileva, Nesmeyanova.     

不同磷脂和糖脂对莱氏衣原体膜上Mg~（2＋）－ATPase活性的影响

莱氏衣原体膜上Ｍｇ~（２＋）－ＡＴＰａｓｅ用ＤＯＣ溶解后,经Ｓｅｐｈａｒｏｓｅ－６Ｂ和ＤＥＡＥ－ＣｅｌｌｕｌｏｓｅＤＥ－５２离子交换柱,得到了部分纯化的Ｍｇ~（２＋）ＡＴＰａｓｅ,并将此ＡＴＰａｓｅ与不同极性头部的磷脂和膜糖脂重组,研究了不同的极性头部的磷脂和膜糖脂对ＡＴＰａｓｅ活性的影响。此酶的活性不依赖酸性磷脂,ＰＧ、ＤＰＧ、大豆磷脂等明显抑制酶活性,中性磷脂ＤＭＰＣ、ＰＥ、ＰＣ则能增加酶活性,其中尤以非双层脂ＰＥ的作用最为明显。从莱氏衣原体膜上提取的糖脂（ＭＧＤＧ,ＤＧＤＧ）单独和ＡＴＰａｓｅ重组时,酶活性增加并不明显,当ＭＧＤＧ和ＤＧＤＧ以等比例混合时,能大大地增加酶活性。这表明Ｍｇ~（２＋）－ＡＴＰａｓｅ的活性很大程度上与磷脂的表面电荷及磷脂的组成相关。     

Dispensable nature of phosphatidylglycerol in Escherichia coli: dual roles of anionic phospholipids

The major anionic phospholipids of Escherichia coli, phosphatidylglycerol (PG) and cardiolipin (CL), have been considered to be indispensable for essential cellular functions, such as the initiation of DNA replication and translocation of proteins across the cytoplasmic membrane. However, we successfully constructed a null pgsA mutant of E. coli that had undetectable levels of PG and CL if the major outer membrane lipoprotein was deficient, clearly indicating that these anionic phospholipids are not indispensable. In the null mutant, we observed the accumulation of phosphatidic acid, an acidic biosynthetic precursor. This suggests a functionally substitutable nature of these anionic phospholipids and allows us to formulate a dual role model for the physiological roles of the anionic phospholipids in E. coli. The anionic phospholipids may play dual roles in E. coli as (i) substrates for head group-specific enzyme reactions, albeit the viability of null PG mutants indicates that the products of head group-specific reactions are not essential; and (ii) those that are replaceable, partly or entirely, by other phospholipids bearing net negative charges, because of their rather loose head group specificity. These two aspects of the physiological roles of anionic phospholipids are discussed with special reference to the phospholipids of other bacteria and eukaryotic organelles.