Phosphatidylinositol synthase from Saccharomyces cerevisiae. Reconstitution, characterization, and regulation of activity

Purified membrane-associated phosphatidylinositol synthase (CDP diacylglycerol:myo-inositol 3-phosphatidyltransferase, EC 2.7.8.11) from Saccharomyces cerevisiae was reconstituted into unilamellar phospholipid vesicles. Reconstitution of the enzyme was performed by removing detergent from an octylglucoside/phospholipid/Triton X-100/enzyme mixed micelle mixture by Sephadex G-50 superfine column chromatography. The average diameter of the vesicles was 40 nm and chymotrypsin treatment of intact vesicles indicated that over 90% of the reconstituted enzyme had its active site facing outward. The enzymological properties and reaction mechanism of reconstituted phosphatidylinositol synthase were determined in the absence of detergent. The reconstituted enzyme was used as a model system to study the regulation of activity. Phosphatidylinositol synthase was constitutive in wild type cells grown in the presence of water-soluble phospholipid precursors as determined by enzyme activity and immunoblotting. Reconstituted enzyme was not effected by water-soluble phospholipid precursors or nucleotides. Maximum activity was found when the enzyme was reconstituted into phosphatidylcholine: phosphatidylethanolamine: phosphatidylinositol: phosphatidylserine vesicles. Phosphatidylserine stimulated reconstituted activity, suggesting that the local phospholipid environment may regulate phosphatidylinositol synthase activity.     

Phospholipid environment alters hormone-sensitivity of the purified insulin receptor kinase.

Insulin receptor kinase, affinity-purified by adsorption and elution from immobilized insulin, is stimulated 2-3-fold by insulin in detergent solution. Reconstitution of the receptor kinase into leaky vesicles containing phosphatidylcholine and phosphatidylethanolamine (1:1, w/w) by detergent removal on Sephadex G-50 results in the complete loss of receptor kinase sensitivity to activation by insulin. Insulin receptors in these vesicles also exhibit an increase in their apparent affinity for 125I-insulin (Kd = 0.12 nM versus 0.76 nM). Inclusion of 8.3-16.7% phosphatidylserine into the reconstituted vesicles restores 40-50% of the insulin-sensitivity to the receptor kinase. An elevated apparent affinity for 125I-insulin of insulin receptors in vesicles containing phosphatidylcholine and phosphatidylethanolamine is also restored to the value observed in detergent solution by the inclusion of phosphatidylserine in the reconstituted system. The effect of phosphatidylserine on insulin receptor kinase appears specific, because cholesterol, phosphatidylinositol and phosphatidic acid are all unable to restore insulin-sensitivity to the receptor kinase. Autophosphorylation sites on the insulin receptor as analysed by h.p.l.c. of tryptic 32P-labelled receptor phosphopeptides are not different for insulin receptors autophosphorylated in detergent solution or for the reconstituted vesicles in the presence or absence of phosphatidylserine. These data indicate that the phospholipid environment of insulin receptors can modulate its binding and kinase activity, and phosphatidylserine acts to restore insulin-sensitivity to the receptor kinase incorporated into phosphatidylcholine/phosphatidylethanolamine vesicles.     

Phospholipid and detergent effects on (Ca2+ + Mg2+)ATPase purified from human erythrocytes

(Ca2+ + Mg2+)ATPase (EC 3.6.1.3) was solubilized from human erythrocyte membranes by detergent extraction with Triton N-101 (0.5 mg/mg membrane protein) and purified by calmodulin affinity chromatography. ATPase activity was assayed in mixtures of Triton N-101 and phospholipid, without reconstitution into bilayer vesicles. At low levels of phospholipid (5 micrograms/ml), the ATPase activity was highly sensitive to the detergent concentration, with maximal activity occurring at or near the critical micelle concentration of the detergent. With increased amounts of phospholipid (50 micrograms/ml), detergent concentrations greater than the critical micelle concentration were required for maximal activity. Detergent alone did not support ATPase activity. Sonicated phospholipid in the form of vesicles was equally ineffective. Activity seemed to be dependent on the presence of detergent/phospholipid mixed micelles. The acidic phospholipids, phosphatidylserine and phosphatidylinositol, as well as the commercial phospholipid preparation, Asolectin, gave activities five to eight times greater than the same amount of phosphatidylcholine. Mixtures of phosphatidylserine and phosphatidylcholine produced intermediate ATPase activities, with the maximal value dependent on the phosphatidylserine concentration. Addition of phosphatidylcholine to fixed concentrations of phosphatidylserine caused a rise in activity that was independent of the ratio of the two phospholipids or the total phospholipid concentration. Phosphatidylcholine may therefore be irreplaceable for some aspect of ATPase function. The number of phospholipid molecules present in mixed micelles at maximal ATPase activity was calculated to be near 50. This value implied that the hydrophobic surface of the ATPase molecule must be completely coated by a single layer of phospholipid molecules for maximum activity to occur.     

Regulation of phospholipid biosynthesis in Saccharomyces cerevisiae by inositol. Inositol is an inhibitor of phosphatidylserine synthase activity

The addition of inositol to the growth medium of Saccharomyces cerevisiae resulted in rapid changes in the rates of phospholipid biosynthesis. The partitioning of the phospholipid intermediate CDP-diacylglycerol was shifted to phosphatidylinositol at the expense of phosphatidylserine and its derivatives phosphatidylethanolamine and phosphatidylcholine. Serine at 133-fold greater concentrations than that of inositol shifted the partitioning of CDP-diacylglycerol to phosphatidylserine at the expense of phosphatidylinositol but to a much lesser degree. Kinetic experiments with pure phosphatidylserine synthase and phosphatidylinositol synthase indicated that the partitioning of CDP-diacylglycerol between phosphatidylserine and phosphatidylinositol was not governed by the affinities both enzymes have for their common substrate CDP-diacylglycerol. Instead, the main regulation of phosphatidylinositol and phosphatidylserine synthesis was through the exogenous supply of inositol. The Km of inositol (0.21 mM) for phosphatidylinositol synthase was 9-fold higher than cytosolic concentration of inositol (24 microM). The Km of serine (0.83 mM) for phosphatidylserine synthase was 3-fold below the cytosolic concentration of serine (2.6 mM). Therefore, inositol supplementation resulted in a dramatic increase in the rate of phosphatidylinositol synthesis, whereas serine supplementation resulted in little affect on the rate of phosphatidylserine synthesis. Inositol also contributed to the regulation of phosphatidylinositol and phosphatidylserine synthesis by having a direct affect on phosphatidylserine synthase activity. Kinetic experiments with pure phosphatidylserine synthase showed that inositol was a noncompetitive inhibitor of the enzyme with a Ki of 65 microM.     

Effect of growth phase on phospholipid biosynthesis in Saccharomyces cerevisiae.

The effect of growth phase on the membrane-associated phospholipid biosynthetic enzymes CDP-diacylglycerol synthase, phosphatidylserine synthase, phosphatidylinositol synthase, and the phospholipid N-methyltransferases in wild-type Saccharomyces cerevisiae was examined. Maximum activities were found in the exponential phase of cells grown in complete synthetic medium. As cells entered the stationary phase of growth, the activities of the CDP-diacylglycerol synthase, phosphatidylserine synthase, and the phospholipid N-methyltransferases decreased 2.5- to 5-fold. The subunit levels of phosphatidylserine synthase and the cytoplasmic-associated enzyme inositol-1-phosphate synthase were not significantly affected by the growth phase. When grown in medium supplemented with inositol-choline, cells in the exponential phase of growth had reduced CDP-diacylglycerol synthase, phosphatidylserine synthase, and phospholipid N-methyltransferase activities, with repressed subunit levels of phosphatidylserine synthase and inositol-1-phosphate synthase compared with cells grown without inositol-choline. Enzyme activity levels remained reduced in the stationary phase of growth of cells supplemented with inositol-choline. The phosphatidylserine synthase and inositol-1-phosphate synthase subunit levels, however, were depressed. Phosphatidylinositol synthase (activity and subunit) was not affected by growth in medium supplemented with or without inositol-choline or the growth phase of the culture. The phospholipid composition of cells in the exponential and stationary phase of growth was also examined. The phosphatidylinositol to phosphatidylserine ratio doubled in stationary-phase cells. The phosphatidylcholine to phosphatidylethanolamine ratio was not significantly affected by the growth phase of cells.     

Regulation of phosphatidylserine synthase from Saccharomyces cerevisiae by phospholipid precursors.

The addition of ethanolamine or choline to inositol-containing growth medium of Saccharomyces cerevisiae wild-type cells resulted in a reduction of membrane-associated phosphatidylserine synthase (CDPdiacylglycerol:L-serine O-phosphatidyltransferase, EC 2.7.8.8) activity in cell extracts. The reduction of activity did not occur when inositol was absent from the growth medium. Under the growth conditions where a reduction of enzyme activity occurred, there was a corresponding qualitative reduction of enzyme subunit as determined by immunoblotting with antiserum raised against purified phosphatidylserine synthase. Water-soluble phospholipid precursors did not effect purified phosphatidylserine synthase activity. Phosphatidylserine synthase (activity and enzyme subunit) was not regulated by the availability of water-soluble phospholipid precursors in S. cerevisiae VAL2C(YEp CHO1) and the opi1 mutant. VAL2C(YEp CHO1) is a plasmid-bearing strain that over produces phosphatidylserine synthase activity, and the opi1 mutant is an inositol biosynthesis regulatory mutant. The results of this study suggest that the regulation of phosphatidylserine synthase by the availability of phospholipid precursors occurs at the level of enzyme formation and not at the enzyme activity level. Furthermore, the regulation of phosphatidylserine synthase is coupled to inositol synthesis.     

Regulation of yeast phosphatidylserine synthase and phosphatidylinositol synthase activities by phospholipids in Triton X-100/phospholipid mixed micelles

The regulation of purified yeast membrane-associated phosphatidylserine synthase (CDP-diacylglycerol:L-serine O-phosphatidyltransferase, EC 2.7.8.8) and phosphatidylinositol synthase (CDP-diacylglycerol:myo-inositol 3-phosphatidyltransferase, EC 2.7.8.11) activities by phospholipids was examined using Triton X-100/phospholipid mixed micelles. Phosphatidate, phosphatidylcholine, and phosphatidylinositol stimulated phosphatidylserine synthase activity, whereas cardiolipin and the neutral lipid diacylglycerol inhibited enzyme activity. Phosphatidate was a potent activator of phosphatidylserine synthase activity with an apparent activation constant (0.033 mol %) 88-fold lower than the apparent Km (2.9 mol %) for the surface concentration of CDP-diacylglycerol. Phosphatidate caused an increase in the apparent Vmax and a decrease in the apparent Km for the enzyme with respect to the surface concentration of CDP-diacylglycerol. Phosphatidylcholine and phosphatidylinositol caused an increase in the apparent Vmax for phosphatidylserine synthase with respect to CDP-diacylglycerol with apparent activation constants of 3.4 and 3.2 mol %, respectively. Cardiolipin and diacylglycerol were competitive inhibitors of phosphatidylserine synthase activity with respect to CDP-diacylglycerol. The apparent Ki value for cardiolipin (0.7 mol %) was 4-fold lower than the apparent Km for CDP-diacylglycerol, whereas the apparent Ki for diacylglycerol (7 mol %) was 2.4-fold higher than the apparent Km for CDP-diacylglycerol. Phosphatidylethanolamine and phosphatidylglycerol did not affect phosphatidylserine synthase activity. Phosphatidylinositol synthase activity was not significantly effected by lipids. The role of lipid activators and inhibitors on phosphatidylserine synthase activity is discussed in relation to overall lipid metabolism.     

Reconstituted phosphatidylserine synthase from Escherichia coli is activated by anionic phospholipids and micelle-forming amphiphiles.

The activity of phosphatidylserine (PS) synthase (CDP-1, 2-diacyl-sn-glycerol: l-serine O-phosphatidyltransferase, EC 2.7.8. 8) from Escherichia coli was studied after reconstitution with lipid vesicles of various compositions. PS synthase exhibited practically no activity in the absence of a detergent and with the substrate CDP-diacylglycerol (CDP-DAG) present only in the lipid vesicles. Inclusion of octylglucoside (OG) in the assay mixture increased the activity 20- to 1000-fold, the degree of activation depending on the lipid composition of the vesicles. Inclusion of additional CDP-DAG in the assay mixture increased the activity 5- to 25-fold. When the fraction of phosphatidylglycerol (PG) was increased from 15 to 100 mol% in the vesicles the activity increased 10-fold using the assay mixture containing OG. The highest activities were exhibited with the anionic lipids synthesized by E. coli, namely PG, diphosphatidylglycerol (DPG), and phosphatidic acid, while phosphatidylinositol gave a lower activity. Cryotransmission electron microscopy showed that transformation of the vesicles to micelles brings about an activation of the enzyme that is proportional to the degree of micellization. Thus, the activity of PS synthase is modulated by the lipid aggregate structure and by the fraction and type of anionic phospholipid in the aggregates. The increase in the activity caused by PG and DPG is physiologically relevant; it may be part of a regulatory mechanism that keeps the balance between phosphatidylethanolamine, and the sum of PG and DPG, nearly constant in wild-type E. coli cells.     

Phospholipid synthesis and lipid composition of subcellular membranes in the unicellular eukaryote Saccharomyces cerevisiae

Subcellular membranes of Saccharomyces cerevisiae, including mitochondria, microsomes, plasma membranes, secretory vesicles, vacuoles, nuclear membranes, peroxisomes, and lipid particles, were isolated by improved procedures and analyzed for their lipid composition and their capacity to synthesize phospholipids and to catalyze sterol delta 24-methylation. The microsomal fraction is heterogeneous in terms of density and classical microsomal marker proteins and also with respect to the distribution of phospholipid-synthesizing enzymes. The specific activity of phosphatidylserine synthase was highest in a microsomal subfraction which was distinct from heavier microsomes harboring phosphatidylinositol synthase and the phospholipid N-methyltransferases. The exclusive location of phosphatidylserine decarboxylase in mitochondria was confirmed. CDO-diacylglycerol synthase activity was found both in mitochondria and in microsomal membranes. Highest specific activities of glycerol-3-phosphate acyltransferase and sterol delta 24-methyltransferase were observed in the lipid particle fraction. Nuclear and plasma membranes, vacuoles, and peroxisomes contain only marginal activities of the lipid-synthesizing enzymes analyzed. The plasma membrane and secretory vesicles are enriched in ergosterol and in phosphatidylserine. Lipid particles are characterized by their high content of ergosteryl esters. The rigidity of the plasma membrane and of secretory vesicles, determined by measuring fluorescence anisotropy by using trimethylammonium diphenylhexatriene as a probe, can be attributed to the high content of ergosterol.     

Reconstituted phosphatidylserine synthase from Escherichia coli is activated by anionic phospholipids and micelle-forming amphiphiles

The activity of phosphatidylserine (PS) synthase (CDP-1,2-diacyl-sn-glycerol: l-serine O-phosphatidyltransferase, EC 2.7.8.8) from Escherichia coli was studied after reconstitution with lipid vesicles of various compositions. PS synthase exhibited practically no activity in the absence of a detergent and with the substrate CDP-diacylglycerol (CDP-DAG) present only in the lipid vesicles. Inclusion of octylglucoside (OG) in the assay mixture increased the activity 20- to 1000-fold, the degree of activation depending on the lipid composition of the vesicles. Inclusion of additional CDP-DAG in the assay mixture increased the activity 5- to 25-fold. When the fraction of phosphatidylglycerol (PG) was increased from 15 to 100 mol% in the vesicles the activity increased 10-fold using the assay mixture containing OG. The highest activities were exhibited with the anionic lipids synthesized by E. coli, namely PG, diphosphatidylglycerol (DPG), and phosphatidic acid, while phosphatidylinositol gave a lower activity. Cryotransmission electron microscopy showed that transformation of the vesicles to micelles brings about an activation of the enzyme that is proportional to the degree of micellization. Thus, the activity of PS synthase is modulated by the lipid aggregate structure and by the fraction and type of anionic phospholipid in the aggregates. The increase in the activity caused by PG and DPG is physiologically relevant; it may be part of a regulatory mechanism that keeps the balance between phosphatidylethanolamine, and the sum of PG and DPG, nearly constant in wild-type E. coli cells.     

Modulation of Tryptophan Hydroxylase Activity by Phospholipids: Stimulation Followed by Inactivation

The activity of tryptophan hydroxylase from the rat brainstem was stimulated rapidly three- to fourfold by the addition of phosphatidylinositol or phosphatidylserine. However, the activity of the enzyme once stimulated was decreased gradually by subsequent incubation with the phospholipid at 37 degrees C, reaching a level below the original activity after 1 h of incubation. The presence of ferrous ion almost perfectly protected the enzyme against this phospholipid inactivation. The activity of the enzyme inactivated by incubation with the phospholipid was not only restored, but also increased further by incubation at 37 degrees C with ferrous ion and dithiothreitol. Gel filtration analysis revealed that the enzyme stimulated by phosphatidylinositol was eluted in a void volume together with the phospholipid vesicles, but the enzyme inactivated by incubation with phosphatidylinositol was eluted at a later region apart from the vesicles. These results, taken together, suggest the possible involvement of cellular membranes in the regulation of tryptophan hydroxylase in the central nervous system.     

Enzymatic detection of phospholipid biosynthetic enzymes after electroblotting.

The membrane-associated enzymes phosphatidylinositol synthase (CDPdiacylglycerol:myo-inositol 3-phosphatidyltransferase; EC 2.7.8.11) and phosphatidylserine synthase (CDPdiacylglycerol:L-serine O-phosphatidyltransferase; EC 2.7.8.8) from Saccharomyces cerevisiae were detected enzymatically after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotting. Enzyme activities were measured on nitrocellulose blots by using pure enzyme preparations as well as Triton X-100-solubilized membranes. Phosphatidylinositol synthase activity migrated to Mr 34,000, and phosphatidylserine synthase activity migrated to Mr 23,000.     

Regulation of phospholipid synthesis in phosphatidylserine synthase-deficient (chol) mutants of Saccharomyces cerevisiae.

chol mutants of Saccharomyces cerevisiae are deficient in the synthesis of the phospholipid phosphatidylserine owing to lowered activity of the membrane-associated enzyme phosphatidylserine synthase. chol mutants are auxotrophic for ethanolamine or choline and, in the absence of these supplements, cannot synthesize phosphatidylethanolamine or phosphatidylcholine (PC). We exploited these characteristics of the chol mutants to examine the regulation of phospholipid metabolism in S. cerevisiae. Macromolecular synthesis and phospholipid metabolism were examined in chol cells starved for ethanolamine. As expected, when chol mutants were starved for ethanolamine, the rates of synthesis of the phospholipids phosphatidylethanolamine and PC declined rapidly. Surprisingly, however, coupled to the decline in PC biosynthesis was a simultaneous decrease in the overall rate of phospholipid synthesis. In particular, the rate of synthesis of phosphatidylinositol decreased in parallel with the decline in PC biosynthesis. The results obtained suggest that the slowing of PC biosynthesis in ethanolamine-starved chol cells leads to a coordinated decrease in the synthesis of all phospholipids. However, under conditions of ethanolamine deprivation in chol cells, the cytoplasmic enzyme inositol-1-phosphate synthase could not be repressed by exogenous inositol, and the endogenous synthesis of the phospholipid precursor inositol appeared to be elevated. The implications of these findings with respect to the coordinated regulation of phospholipid synthesis are discussed.     

Properties of particulate and solubilized phosphatidylserine synthase activity from Saccharomyces cerevisiae. Inhibitory effect of choline in the growth medium

When radiolabeled serine is incubated with a particulate fraction from Saccharomyces cerevisiae, radioactivity is incorporated initially into phosphatidylserine and gradually appears in phosphatidylethanolamine. Because decarboxylation of phosphatidylserine is blocked by hydroxylamine, phosphatidylserine synthase can be assayed separately. The yeast phosphatidylserine synthase activity 1) exhibits a divalent cation requirement; 2) is stimulated by exogenous CDP-diolein (apparent Km = 0.17 mM); 3) has an apparent Km = 4 mM for L-serine; 4) has a neutral pH optimum; 5) is inhibited by p-hydroxymercuribenzoate; and 6) is reversible in the presence of 5'-CMP, but not 2'-CMP, 3'-CMP, or 5'-AMP. The phospholipid-synthesizing activity is solubilized with Triton X-100 and the enzymatic parameters have been compared with the particulate form of the enzyme. Detergent extracts catalyze the conversion of exogenous purified [31P]CDP-diglyceride to [32P]phosphatidylserine in the presence of Mn2+ and L-serine. Enzyme preparations from cells grown in the presence of choline, that have reduced phospholipid methylation activity (Waechter, C. J., Steiner, M. R., and Lester, R. L. (1969) J. Biol. Chem. 244, 3419-3422), also have substantially less phosphatidylserine synthase activity compared to identical preparations grown in the absence of choline. When choline, phosphocholine, CDP-choline, and phosphatidylcholine are present in vitro, there is no direct inhibitory effect on phosphatidylserine synthase activity. While the inclusion of choline in the growth medium caused a significant reduction in phosphatidylserine synthase activity, it did not appreciably effect the apparent Km values for L-serine and CDP-diglyceride. These results are consistent with choline-grown cells containing less phosphatidylserine synthase activity because of lower amounts of enzyme present or perhaps less active enzyme due to covalent modification.     

Incorporation of a bacterial membrane-bound hydrogenase into proteoliposomes.

Membrane-bound nickel-iron hydrogenases from diverse genera of bacteria have been previously characterized and they are closely related. We report the reconstitution of purified Bradyrhizobium japonicum hydrogenase into proteoliposomes by a detergent dialysis method followed by two or three cycles of freeze-thaw. Sedimentation experiments revealed that more than 60% of the H2-uptake activity was particulate when reconstituted into Escherichia coli phospholipids. Sucrose-gradient centrifugation separated hydrogenase activity into two peaks, the less dense of which was phospholipid-associated and turbid, thereby showing successful incorporation. Purified enzyme did not bind to performed phospholipid vesicles, and 1.0 M NaCl failed to remove incorporated hydrogenase. The optimal micellar detergent:phospholipid ratio (rho) value for hydrogenase incorporation was 2.0. Proteoliposomes containing acidic phospholipids were the most effective for incorporation as well as for activity. The artificial electron acceptor specificity was similar for proteoliposomes and for H2-oxidizing membranes from B. japonicum. Proteoliposomes formed under optimal conditions had a broad size distribution centered around 400 nm diameter. Hydrogenase activity in proteoliposomes was partially protected from inactivation by the protein modification reagent diazobenzene sulfonate (DABS) (inactivation t1/2 = 30 min), whereas DABS rapidly inactivated the purified enzyme (t1/2 = 4 min). The latter result indicates protection of a catalytically important site by the phospholipid bilayer. This experimental system should be useful in addressing questions regarding the in vivo situation of bacterial membrane-bound hydrogenases.     

Regulation of phospholipid biosynthesis in Saccharomyces cerevisiae by cyclic AMP-dependent protein kinase.

The addition of cyclic AMP (cAMP) to Saccharomyces cerevisiae cyr1 mutant cells resulted in an increase in the rate of phosphatidylinositol synthesis at the expense of phosphatidylserine synthesis. The decrease in phosphatidylserine synthesis correlated with the down regulation of phosphatidylserine synthase activity by cAMP-dependent protein kinase phosphorylation. The increase in phosphatidylinositol synthesis was not due to the regulation of phosphatidylinositol synthase by cAMP-dependent protein kinase.     

Reconstitution of the skeletal sarcoplasmic reticulum Ca2(+)-pump: influence of negatively charged phospholipids.

The Ca2(+)-ATPase of skeletal sarcoplasmic reticulum was purified and reconstituted in the presence of phosphatidyl choline using the freeze-thaw sonication technique. The effect of incorporation of negatively charged phospholipids, phosphatidylserine and phosphatidylinositol phosphate, into the phosphatidylcholine proteoliposomes was investigated. Various ratios of phosphatidylserine or phosphatidylinositol phosphate to phosphatidylcholine were used, while the total amount of phospholipid in the reconstituted vesicles was kept constant. Enrichment of phosphatidylcholine proteoliposomes by phosphatidylserine or phosphatidylinositol phosphate was associated with activation of Ca2(+)-uptake and Ca2(+)-ATPase activities. The highest activation was obtained at a 50:50 molar ratio of phosphatidylserine:phosphatidylcholine and at a 10:90 molar ratio of phosphatidylinositol phosphate:phosphatidylcholine. The initial rates of Ca2(+)-uptake obtained at 1 microM Ca2+ were 2.6 +/- 0.1 mumol/min per mg of phosphatidylserine:phosphatidylcholine proteoliposomes and 1.5 +/- 0.1 mumol/min per mg of phosphatidylinositol phosphate:phosphatidylcholine proteoliposomes, compared to 0.9 +/- 0.05 mumol/min per mg of phosphatidylcholine proteoliposomes. These findings suggest that negatively charged phospholipids may be involved in the activation of the reconstituted skeletal muscle sarcoplasmic reticulum Ca2(+)-pump.     

Isolation and characterization of highly purified mitochondrial outer membranes of the yeast Saccharomyces cerevisiae (Method)

Mitochondrial outer membrane vesicles (OMV) from the yeast Saccharomyces cerevisiae were prepared by osmotic swelling and mechanical disruption of mitochondria that had been isolated at pH 6.0 and purified by density gradient centrifugation. The OMV were obtained in a yield of 1% (protein/protein) with respect to the mitochondria. The OMV were shown to be essentially free of mitochondrial inner membrane protein markers, while contamination with endoplasmic reticulum was around 5% (protein-based). The very low phosphatidylserine synthase activity present in the OMV preparation indicated that contamination with mitochondria-associated membranes (MAM) was negligible. The resistance of the outer membrane protein Tom40 to digestion by trypsin demonstrated the sealed nature and right-side out orientation of the OMV. Analysis of the phospholipid composition revealed that the contents of phosphatidylcholine and phosphatidylinositol are higher and the content of phosphatidylethanolamine is lower in the mitochondrial outer membrane as compared to whole mitochondria. Cardiolipin is largely depleted in the OMV.     

Isolation and characterization of highly purified mitochondrial outer membranes of the yeast Saccharomyces cerevisiae (method).

Mitochondrial outer membrane vesicles (OMV) from the yeast Saccharomyces cerevisiae were prepared by osmotic swelling and mechanical disruption of mitochondria that had been isolated at pH 6.0 and purified by density gradient centrifugation. The OMV were obtained in a yield of 1% (protein/protein) with respect to the mitochondria. The OMV were shown to be essentially free of mitochondrial inner membrane protein markers, while contamination with endoplasmic reticulum was around 5% (protein-based). The very low phosphatidylserine synthase activity present in the OMV preparation indicated that contamination with mitochondria-associated membranes (MAM) was negligible. The resistance of the outer membrane protein Tom40 to digestion by trypsin demonstrated the sealed nature and right-side out orientation of the OMV. Analysis of the phospholipid composition revealed that the contents of phosphatidylcholine and phosphatidylinositol are higher and the content of phosphatidylethanolamine is lower in the mitochondrial outer membrane as compared to whole mitochondria. Cardiolipin is largely depleted in the OMV.