On the mechanism of the mitochondrial decarboxylation of phosphatidylserine.

To study intramitochondrial phospholipid flow, radiolabeled phosphatidylserine was introduced into isolated rat liver mitochondria from donor vesicles through the action of a nonspecific lipid transfer protein. Imported phosphatidylserine was rapidly decarboxylated to phosphatidylethanolamine. Both the imported phosphatidylserine and the formed phosphatidylethanolamine were confined to the outer membrane. The enzyme phosphatidylserine decarboxylase was shown to be located exclusively in the inner membrane. It was not enriched in isolated contact site fractions. 1,4-Dinitrophenol caused an inhibition of the decarboxylation of phosphatidylserine. This inhibition was not due to the uncoupling of the oxidative phosphorylation itself, but possibly due to a decrease in the number of contact sites. This suggests that phosphatidylserine flows from the outer membrane to the inner membrane through contact sites between inner and outer membrane to become decarboxylated and that the formed phosphatidylethanolamine flows directly back to the outer membrane, without mixing with inner membrane phosphatidylethanolamine.     

Subcellular and submitochondrial localization of phospholipid-synthesizing enzymes in Saccharomyces cerevisiae.

Using highly enriched membrane preparations from lactate-grown Saccharomyces cerevisiae cells, the subcellular and submitochondrial location of eight enzymes involved in the biosynthesis of phospholipids was determined. Phosphatidylserine decarboxylase and phosphatidylglycerolphosphate synthase were localized exclusively in the inner mitochondrial membrane, while phosphatidylethanolamine methyltransferase activity was confined to microsomal fractions. The other five enzymes tested in this study were common both to the outer mitochondrial membrane and to microsomes. The transmembrane orientation of the mitochondrial enzymes was investigated by protease digestion of intact mitochondria and of outside-out sealed vesicles of the outer mitochondrial membrane. Glycerolphosphate acyltransferase, phosphatidylinositol synthase, and phosphatidylserine synthase were exposed at the cytosolic surface of the outer mitochondrial membrane. Cholinephosphotransferase was apparently located at the inner aspect or within the outer mitochondrial membrane. Phosphatidate cytidylyltransferase was localized in the endoplasmic reticulum, on the cytoplasmic side of the outer mitochondrial membrane, and in the inner mitochondrial membrane. Inner membrane activity of this enzyme constituted 80% of total mitochondrial activity; inactivation by trypsin digestion was observed only after preincubation of membranes with detergent (0.1% Triton X-100). Total activity of those enzymes that are common to mitochondria and the endoplasmic reticulum was about equally distributed between the two organelles. Data concerning susceptibility to various inhibitors, heat sensitivity, and the pH optima indicate that there is a close similarity of the mitochondrial and microsomal enzymes that catalyze the same reaction.     

The enzymes of phospholipid synthesis in Clostridium butyricum

We have examined extracts of Clostridium butyricum for several enzymes of phospholipid synthesis. Membrane particles were shown to catalyze the formation of CDP-diglyceride from [3H]CTP and phosphatidic acid. The reaction was dependent on Mg2+ and stimulated by monovalent cations. CDP-diglyceride formed in vitro was found to be a substrate for both phosphatidylglycerophosphate synthetase and phosphatidylserine synthetase. The formation of phosphatidylglycerophosphate from added CDP-diglyceride and [U-14C]sn-glycerol-3-phosphate was dependent on Mg2+ and Triton X-100. The dephosphorylation of endogenously-generated phosphatidylglycerophosphate to yield phosphatidylglycerol was observed to be pH-dependent. The formation of phosphatidylserine from CDP-diglyceride and L-[3-14C]serine was stimulated by Mg2+ and Triton X-100. dCDP-diglyceride was a suitable substrate for both phosphatidylglycerophosphate synthetase and phosphatidylserine synthetase. Phosphatidylserine decarboxylase activity was barely detectable in membrane particles from C. butyricum. The addition of E. coli membrane particles provided efficient phosphatidylserine decarboxylase activity in this system. Although plasmalogens are the principal lipids of C. butyricum, none of the products of phospholipid synthesis formed in vitro contained measurable amounts of plasmalogens. The subcellular distribution of both phosphatidylglycerophosphate synthetase and phosphatidylserine synthetase in C. butyricum was also studied. Both were found to be membrane-associated.     

Intramitochondrial transfer of phospholipids in the yeast, Saccharomyces cerevisiae

Translocation of phosphatidylinositol, which is synthesized on the outer aspect of the outer membrane of isolated yeast mitochondria, to the inner membrane is linked to phosphatidylinositol synthesis and is therefore a vectorial process. Phosphatidylinositol once integrated into the inner mitochondrial membrane is not transferred back to the mitochondrial surface. Phosphatidylserine is also translocated from the outer to the inner mitochondrial membrane, where it is decarboxylated to phosphatidylethanolamine. We made use of this metabolic modification to characterize the intramitochondrial transfer of phosphatidylserine and phosphatidylethanolamine. Intramitochondrial phosphatidylserine transfer is insensitive to the uncoupler carbonyl cyanide m-chlorophenylhydrazone and to valinomycin and is thus independent of an electrochemical gradient across the inner membrane. Transfer of phosphatidylserine from the outer to the inner mitochondrial membrane occurs not only in intact mitochondria but also in mitoplasts which are devoid of intermembrane space proteins but have the outer membrane still adherent to the inner membrane. This result suggests that specific contact sites are involved in the intramitochondrial translocation of phospholipids. 3H-Labeled phosphatidylethanolamine synthesized from [3H]serine in isolated mitochondria is readily exported from the inner to the outer mitochondrial membrane without prior mixing with the pool of phosphatidylethanolamine of the inner membrane.     

Reconstitution of phosphatidylserine import into rat liver mitochondria

The synthesis translocation and decarboxylation of phosphatidylserine occurs in a cell-free system. The principal membrane components necessary are microsomes (source of phosphatidylserine synthase) and mitochondria (source of phosphatidylserine decarboxylase). The interorganelle translocation of phosphatidylserine can be measured by quantitating the decarboxylation of phosphatidyl[1'-14C]serine initially present in prelabeled microsomal membranes using a 14CO2 trapping assay. The decarboxylation of microsomal phosphatidylserine by intact mitochondria is 1) dependent upon substrate (microsomal membrane) concentration, 2) different from decarboxylation of liposomal phosphatidylserine, 3) resistant to proteases, 4) independent of soluble factors, and 5) unaffected by the addition of partially purified phospholipid exchange proteins but accelerated by purified nonspecific phospholipid exchange protein. The rate-limiting step in the reconstituted translocation-decarboxylation system is not the decarboxylation reaction but the initial translocation event between the microsomal membrane and the outer mitochondrial membrane. These data are interpreted to demonstrate that phosphatidylserine import into the mitochondria can occur via collision complexes formed between the endoplasmic reticulum or vesicles derived therefrom and the outer mitochondrial membrane.     

Specific interaction of protein tyrosine phosphatase-MEG2 with phosphatidylserine

Protein tyrosine phosphatase (PTP)-MEG2 is an intracellular tyrosine phosphatase that contains a Sec14 homology domain. We have purified the full-length and truncated forms of the enzyme from recombinant adenovirus-infected human 293 cells. By using lipid-membrane overlay and liposome binding assays, we demonstrated that PTP-MEG2 specifically binds phosphatidylserine among over 20 lipid compounds tested. The binding is mediated by its N-terminal Sec14 domain. In intact cells, the Sec14 domain is responsible for localization of PTP-MEG2 to the perinuclear region, and uploading of PS into the cell membrane causes translocation of PTP-MEG2 to the plasma membrane. Phosphatidylserine is a relatively abundant cell membrane phospholipid non-symmetrically distributed in the outer layer and inner layer of cell membranes. It has recently been defined as an important ligand for clearance of apoptotic cells. By specifically binding phosphatidylserine, PTP-MEG2 may play an important role in regulating signaling processes associated with phagocytosis of apoptotic cells.     

Identification of bound pyruvate essential for the activity of phosphatidylserine decarboxylase of Escherichia coli.

Phosphatidylserine decarboxylase, an intrinsic membrane protein of Escherichia coli, catalyzes the decarboxylation of phosphatidylserine, the final step in the biosynthesis of phosphatidylethanolamine, the principal membrane lipid of this organism. The purified enzyme lacks the absorption spectrum characteristic of pyridoxal-containing enzymes, and it has now been found to contain bound pyruvate, the carbonyl function of which is essential for catalytic activity. The decarboxylase is inactivated by treatment with a number of reagents that attack carbonyl groups, including sodium borohydride. Reduction with tritiated borohydride leads to the introduction of stably bound radioactivity, which, after acid hydrolysis, has been identified as tritiated lactate by several chromatographic procedures and by treatment with lactate dehydrogenase. The enzyme resists inactivation by cyanoborohydride in the absence of substrate, but is readily inactivated by this reagent in the presence of phosphatidylserine. Under the conditions of treatment of neutral pH, cyanoborohydride does not react with carbonyl residues at an appreciable rate, but reduces imino groups much more rapidly. This finding, together with demonstrated dependence of the enzyme upon the carbonyl residue of pyruvate for activity, strongly suggests that a Schiff base is formed by addition of the amino group of phosphatidylserine to the pyruvate residue of the enzyme as an essential step in the action of the decarboxylase.     

Translocation of phospholipids between the outer and inner membranes of Salmonella typhimurium.

The reversibility and specificity of phospholipid translocation between the inner and outer membrane of Salmonella typhimurium has been investigated by incorporating exogenous lipids from phospholipid vesicles into the outer membrane of intact cells. Translocation of newly incorporated phospholipids to the inner membrane was demonstrated by decarboxylation of vesicle-derived phosphatidylserine and by recovery of vesicle constituents in both inner and outer membrane fractions. All Salmonella phospholipids tested, as well as phosphatidylcholine and cholesteryl oleate were effectively translocated to the inner membrane. However, no translocation of vesicle-derived lipopolysaccharide or an incomplete biosynthetic precursor of lipid A could be detected. Translocation of phospholipids and cholesteryl ester was rapid and extensive, and appeared to lead to equilibration of the lipids between the two membranes. The mechanism of intermembrane translocation has not been established, but the results are suggestive of diffusional flow across zones of adhesion between the inner and outer membranes.     

Mitochondrial membrane contact sites of yeast. Characterization of lipid components and possible involvement in intramitochondrial translocation of phospholipids

Submitochondrial membrane fractions from yeast that are enriched in inner and outer membrane contact sites were analyzed with respect to their lipid composition. Characteristic features were the significantly reduced content of phosphatidylinositol, the decreased amount of phosphatidylcholine, and the enrichment in phosphatidylethanolamine and cardiolipin. Coisolation of phosphatidylserine synthase with the outer membrane portion and enrichment of phosphatidylserine decarboxylase in the inner membrane portion of isolated contact sites provided the basis for a metabolic assay to study phosphatidylserine transfer from the outer to the inner mitochondrial membrane via contact sites. The efficient conversion to [3H]phosphatidylethanolamine of [3H]phosphatidylserine synthesized from [3H]serine in situ supports the notion that mitochondrial membrane contact sites are zones of intramitochondrial translocation of phosphatidylserine.     

Involvement of contact sites in phosphatidylserine import into liver mitochondria

The synthesis, translocation, and decarboxylation of phosphatidylserine can occur in a cell-free system (Voelker, D. R. (1989) J. Biol. Chem. 264, 8019-8025). We made use of the spatial separation of the site of biosynthesis and the site of decarboxylation of phosphatidylserine to demonstrate that mitochondrial contact sites are intimately involved in the translocation of phosphatidylserine prior to decarboxylation. In that sense, the inhibition of phosphatidylserine decarboxylase leads to an accumulation of this phospholipid in the contact site-enriched fractions without mixing the inner membrane phospholipid pool. On the other hand, newly synthesized phosphatidylethanolamine can be exported very rapidly to the mitochondrial surface in the same way, i.e. via contact sites. These data provide further evidence for the existence of a structural and functional microcompartmentation at the inner mitochondrial membrane surface.     

Changes in the phospholipid composition and phospholipid asymmetry of ram sperm plasma membranes after cryopreservation

The changes in the phospholipid composition of spermatozoa plasma membranes after freezing were determined by thin-layer chromatography. The results showed an augmentation of the diphosphatidylglycerol and a diminution of phosphatidylglycerol, phosphatidylserine, and phosphatidylethanolamine in sperm plasma membranes after freezing. In intact sperm cells we observed an elevation of the sphingomyelin and phosphatidylinositol levels and a diminution of the phosphatidylethanolamine and diphosphatidylglycerol levels. The effect of freezing on the phospholipid distribution between the inner and outer monolayers of the plasma membrane was also studied using exogenous phospholipases and trinitrobenzene sulfonate. The most important change we observed after freezing, was the translocation of diphosphatidylglycerol from the inner to the outer monolayer of the plasma membrane.     

Relationship between phospholipid compositions and transport activities of amino acids in Escherichia coli membrane vesicles.

Cells of Escherichia coli were incubated in broth medium in the presence of 5 mM of hydroxylamine which completely inhibited growth but did not affect viabilities. Hydroxylamine is known to inhibit phosphatidylserine decarboxylase. A large amount of phosphatidylserine (up to 20% of total phospholipids), which did not occur in normal cells, accumulated accompanied with a decrease in phosphatidylethanolamine. Higher uptake activities of serine and glutamate were observed with the hydroxylamine-treated cells than control cells. When membrane vesicles from hydroxylamine-treated cells were prepared, they also displayed higher uptake activities of serine, proline, glutamate, and threonine than those of normal membranes. When hydroxylamine-treated cells were incubated with chloramphenicol, at concentrations which almost completely inhibited protein synthesis, the composition of phosphatidylserine decreased with a concomitant increase in that of phosphatidylethanolamine. The phospholipid composition of these cells incubated for 5 h with chloramphenicol became almost normal. Membranes vesicles prepared from such cells displayed reduced uptake activities, which were close to those of normal vesicles. These results were interpreted as indicating the altered transport activities due to the altered phospholipid composition.     

Effect of erythrocyte transbilayer phospholipid distribution on fusion with vesicular stomatitis virus

To identify the specific component(s) in the target membrane involved in fusion of vesicular stomatitis virus (VSV), we examined the interaction of the virus with human erythrocyte membranes with asymmetric and symmetric bilayer distributions of phospholipids. Fusion was monitored spectrofluorometrically by the octadecylrhodamine dequenching assay. Fusion of VSV with lipid-symmetric erythrocyte ghosts was rapid at 37 degrees C and low pH, whereas little or no fusion was observed with lipid-asymmetric ghosts. Conversion of phosphatidylserine in the lipid-symmetric ghost membrane to phosphatidylethanolamine by means of the enzyme phosphatidylserine decarboxylase did not alter the target membrane's susceptibility to VSV fusion. Spin-labeled phospholipid analogues with phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine headgroups incorporated into the outer leaflet of lipid-asymmetric erythrocytes did not render those membranes fusogenic. Electron spin resonance spectra showed an increased mobility of a phosphatidylcholine spin-label incorporated into the outer leaflet of lipid-symmetric erythrocyte ghosts as compared to that of lipid-asymmetric ghosts. These results indicate that the susceptibility to VSV fusion is not dependent on any particular phospholipid but rather is related to packing characteristics of the target membrane.     

Disruption of phosphatidylserine translocation to the mitochondria in baby hamster kidney cells

Phosphatidylserine synthase is found predominantly in the microsomal fraction, and phosphatidylserine decarboxylase is found predominantly in the mitochondrial fraction of baby hamster kidney (BHK-21) cells. This segregation of enzymes of phosphatidylserine metabolism allows serine metabolism to phosphatidylserine and phosphatidylethanolamine to be used as an indicator of the intracellular movement of phosphatidylserine. After BHK-21 cells were pulse-labeled with [3H]serine, phosphatidylserine was efficiently labeled, and subsequently 40-50% of this radiolabeled lipid turned over to form phosphatidylethanolamine during a 7.5-h chase. Treatment of cells with NaN3 plus NaF or cycloheximide at the end of the pulse labeling period markedly inhibited the rate and extent of phosphatidylserine turnover during the chase period. The inhibition of phosphatidylserine turnover could not be attributed to inhibition of either phosphatidylserine decarboxylase or phosphatidylserine exchange protein activity. Subcellular fractionation of the BHK-21 cells demonstrated that cells poisoned with NaN3 plus NaF accumulated phosphatidylserine in the microsomal fraction relative to unpoisoned cells. The results indicate that metabolic energy is required for the transport of phosphatidylserine to the mitochondria.     

Phospholipid localization in the plasma membrane of Friend erythroleukemic cells and mouse erythrocytes

The distribution of phospholipids over outer and inner layers of the plasma membranes of Friend erythroleukemic cells (Friend cells) and mature mouse erythrocytes has been determined. The various techniques which have been applied to establish the phospholipid localization include the following: phospholipase A2, phospholipase C, and sphingomyelinase C treatment, fluorescamine labeling of phosphatidylethanolamine, and a phosphatidylcholine transfer protein mediated exchange procedure. The data obtained with these different techniques were found to be in good agreement with each other. Phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol were found to be distributed symmetrically over both layers of the plasma membrane of Friend cells. In contrast, sphingomyelin was found to be enriched in the outer layer of the membrane (80-85%), and phosphatidylserine appeared to be present mainly in the inner layer (80-90%). From these results, it was calculated that the outer and inner layers accounted for 46% and 54%, respectively, of the total phospholipid complement of that membrane. Analogous studies on the plasma membrane of mature mouse erythrocytes showed that the transbilayer distribution of the total phospholipid mass appeared to be the same as in the plasma membrane of the Friend cell, namely, 46% and 54% in outer and inner layers, respectively. The outer layer of this membrane contains 57% of the phosphatidylcholine, 20% of the phosphatidylethanolamine, 85% of the sphingomyelin, and 42% of the phosphatidylinositol, and none of the phosphatidylserine was present.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Transport of Phosphatidylserine from Microsomes to the Inner Mitochondrial Membrane in Brain Tissue

Abstract: Phosphatidylserine was labeled by incubating rat brain homogenates with [3-¹⁴C]serine in the presence of Ca²⁺ (base-exchange conditions). Some labeled phosphati-dylethanolamine also forms, in spite of the inhibition of Ca²⁺ on phosphatidylserine decarboxylase. Phosphatidylserine labeling and decarboxylation also occur on incubating a mixture of purified mitochondria and microsomes, suggesting that no soluble factors are necessary for the synthesis and the decarboxylation of phosphatidylserine. Ca²⁺ favors the transfer of phosphatidylserine from microsomes (where it forms) to mitochondria (where it is decarboxylated). The specific radioactivity of the phosphatidylserine transferred to mitochondria is higher than that of microsomal phosphatidylserine. This finding supports the hypothesis that the lipid is compartmentalized in microsomes and that radioactive, newly synthesized phosphatidylserine is much better exported than the bulk of microsomal phospholipid.     

Phospholipid asymmetry in the isolated sarcoplasmic reticulum membrane

The total phospholipid content and distribution of phospholipid species between the outer and inner monolayers of the isolated sarcoplasmic reticulum membrane was measured by phospholipase A2 activities and neutron diffraction. Phospholipase measurements showed that specific phospholipid species were asymmetric in their distribution between the outer and inner monolayers of the sarcoplasmic reticulum lipid bilayer; phosphatidylcholine (PC) was distributed 48/52 +/- 2% between the outer and inner monolayer of the sarcoplasmic reticulum bilayer, 69% of the phosphatidyl-ethanolamine (PE) resided mainly in the outer monolayer of the bilayer, 85% of the phosphatidylserine (PS) and 88% of the phosphatidylinositol (PI) were localized predominantly in the inner monolayer. The total phospholipid distribution determined by these measurements was 48/52 +/- 2% for the outer/inner monolayer of the sarcoplasmic reticulum lipid bilayer. Sarcoplasmic reticulum phospholipids were biosynthetically deuterated and exchanged into isolated vesicles with both a specific lecithin and a general exchange protein. Neutron diffraction measurements directly provided lipid distribution profiles for both PC and the total lipid content in the intact sarcoplasmic reticulum membrane. The outer/inner monolayer distribution for PC was 47/53 +/- 1%, in agreement with phospholipase measurements, while that for the total lipid was 46/54 +/- 1%, similar to the phospholipase measurements. These neutron diffraction results regarding the sarcoplasmic reticulum membrane bilayer were used in model calculations for decomposing the electron-density profile structure (10 A resolution) of isolated sarcoplasmic reticulum previously determined by X-ray diffraction into structures for the separate membrane components. These structure studies showed that the protein profile structure within the membrane lipid bilayer was asymmetric, complementary to the asymmetric lipid structure. Thus, the total phospholipid asymmetry obtained by two independent methods was small but consistent with a complementary asymmetric protein structure, and may be related to the highly vectorial functional properties of the calcium pump ATPase protein in the sarcoplasmic reticulum membrane.     

Phospholipid import into mitochondria: possible regulation mediated through lipid polymorphism.

We previously demonstrated that the translocation of microsomal phosphatidylserine to the inner mitochondrial membrane occurs via contact sites before decarboxylation. According to the specific lipid composition of contact sites, we investigated lipid polymorphism as a possible regulation mechanism of phospholipid import into mitochondria. Phosphatidylserine import into mitochondria is increased in the presence of calcium, under conditions where non bilayer lipid-structures can be induced in cardiolipin-containing membranes. The results are discussed in terms of structural as well as functional domains heterogeneity within contact sites.     

The role of phosphatidylserine in recognition of apoptotic cells by phagocytes

Exposure of phosphatidylserine on the outer leaflet of the plasma membrane is a surface change common to many apoptotic cells. Normally restricted to the inner leaflet, phosphatidylserine appears as a result of decreased aminophospholipid translocase activity and activation of a calcium-dependent scramblase. Phosphatidylserine exposure has several potential biological consequences, one of which is recognition and removal of the apoptotic cell by phagocytes. It is still not clear which receptors mediate PS recognition on apoptotic cells; however, several interesting candidates have been proposed. These include the Class B scavenger and thrombospondin receptor CD36, an oxLDL receptor (CD68), CD14, annexins, beta2 glycoprotein I, gas-6 and a novel activity expressed on macrophages stimulated with digestible particles such as beta-glucan. Whether PS is the sole ligand recognized by phagocytes or whether it associated with other molecules to form a complex ligand remains to be determined.