Asymmetry of the site of choline incorporation into phosphatidylcholine of rat liver microsomes.

[14C]Choline was incorporated into microsomal membranes in vivo, and from CDP-[14C]choline in vitro, and the site of incorporation determined by hydrolysis of the outer leaflet of the membrane bilayer using phospholipase C from Clostridium welchii. Labelled phosphatidylcholine was found to be concentrated in the outer leaflet of the membrane bilayer with a specific activity approximately three times that of the inner leaflet. During incorporation of CDP-choline and treatment with phospholipase C the vesicles retained labelled-protein contents indicating that they remained intact. When the microsomes were opened with taurocholate after incorporation of [14C]choline in vivo, the labelled phosphatidylcholine behaved as a single pool. Selective hydrolysis of labelled phosphatidylcholine in intact vesicles is not, therefore, a consequence of specificity of phospholipase C. These results indicate that the phosphatidylcholine of the outer leaflet of the microsomal membrane bilayer is preferentially labelled by the choline-phosphotransferase pathway and that this pool of phospholipid does not equilibrate with that of the inner leaflet.     

Chloroplast biogenesis. Regulation of lipid transport to the thylakoid in chloroplasts isolated from expanding and fully expanded leaves of pea

To study the regulation of lipid transport from the chloroplast envelope to the thylakoid, intact chloroplasts, isolated from fully expanded or still-expanding pea (Pisum sativum) leaves, were incubated with radiolabeled lipid precursors and thylakoid membranes subsequently were isolated. Incubation with UDP[(3)H]Gal labeled monogalactosyldiacylglycerol in both envelope membranes and digalactosyldiacylglycerol in the outer chloroplast envelope. Galactolipid synthesis increased with incubation temperature. Transport to the thylakoid was slow below 12 degrees C, and exhibited a temperature dependency closely resembling that for the previously reported appearance and disappearance of vesicles in the stroma (D.J. Morré, G. Selldén, C. Sundqvist, A.S. Sandelius [1991] Plant Physiol 97: 1558-1564). In mature chloroplasts, monogalactosyldiacylglycerol transport to the thylakoid was up to three times higher than digalactosyldiacylglycerol transport, whereas the difference was markedly lower in developing chloroplasts. Incubation of chloroplasts with [(14)C]acyl-coenzyme A labeled phosphatidylcholine (PC) and free fatty acids in the inner envelope membrane and phosphatidylglycerol at the chloroplast surface. PC and phosphatidylglycerol were preferentially transported to the thylakoid. Analysis of lipid composition revealed that the thylakoid contained approximately 20% of the chloroplast PC. Our results demonstrate that lipids synthesized at the chloroplast surface as well as in the inner envelope membrane are transported to the thylakoid and that lipid sorting is involved in the process. Furthermore, the results also indicate that more than one pathway exists for galactolipid transfer from the chloroplast envelope to the thylakoid.     

Preparation and characterization of membrane fractions enriched in outer and inner envelope membranes from spinach chloroplasts. II. Biochemical characterization

In the previous paper (Block, M. A., Dorne, A.-J., Joyard, J., and Douce, R. (1983) J. Biol. Chem. 258, 13273-13280), we have described a method for the separation of membrane fractions enriched in outer and inner envelope membranes from spinach chloroplasts. The two envelope membranes have a different weight ratio of acyl lipid to protein (2.5-3 for the outer envelope membrane and 0.8-1 for the inner envelope membrane). The two membranes also differ in their polar lipid composition. However, in order to prevent the functioning of the galactolipid:galactolipid galactosyltransferase during the course of envelope membrane separation, we have analyzed the polar lipid composition of each envelope membrane after thermolysin treatment of the intact chloroplasts. The outer envelope membrane is characterized by the presence of high amounts of phosphatidylcholine and digalactosyldiacylglycerol whereas the inner envelope membrane has a polar lipid composition almost identical with that of the thykaloids. No phosphatidylethanolamine or cardiolipin could be detected in either envelope membranes, thus demonstrating that the envelope membranes, and especially the outer membrane, do not resemble extrachloroplastic membranes. No striking differences were found in the fatty acid composition of the polar lipids from either the outer or the inner envelope membrane. The two envelope membranes also differ in their carotenoid composition. Among the different enzymatic activities associated with the chloroplast envelope, we have shown that the Mg2+-dependent ATPase, the UDP-Gal:diacylglycerol galactosyltransferase, the phosphatidic acid phosphatase, and the acyl-CoA thioesterase are associated with the inner envelope from spinach chloroplasts whereas the acyl-CoA synthetase is located on the outer envelope membrane.     

In situ incorporation of Fatty acids into lipids of the outer and inner envelope membranes of pea chloroplasts

When incubated with [1-¹⁴C]acetate and cofactors (ATP, Coenzyme A, sn-glycerol-3-phosphate, UDPgalactose, and NADH), intact chloroplasts synthesized fatty acids that were subsequently incorporated into most of the lipid classes. To study lipid synthesis at the chloroplast envelope membrane level, ¹⁴C-labeled pea (Pisum sativum) chloroplasts were subfractionated using a single flotation gradient. The different envelope membrane fractions were characterized by their density, lipid and polypeptide composition, and the localization of enzymic activities (UDPgalactose-1,2 diacylglycerol galactosyltransferase, Mg²⁺-dependent ATPase). They were identified as very pure outer membranes (light fraction) and strongly enriched inner membranes (heavy fraction). A fraction of intermediate density, which probably contained double membranes, was also isolated. Labeled glycerolipids recovered in the inner envelope membrane were phosphatidic acid, phosphatidyl-glycerol, 1,2 diacylglycerol, and monogalactosyldiacylglycerol. Their ¹⁴C-fatty acid composition indicated that a biosynthetic pathway similar to the prokaryotic pathway present in cyanobacteria occurred in the inner membrane. In the outer membrane, phosphatidylcholine was the most labeled glycerolipid. Phosphatidic acid, phosphatidylglycerol, 1,2 diacylglycerol, and monogalactosyldiacylglycerol were also labeled. The ¹⁴C-fatty acid composition of these lipids showed a higher proportion of oleate than palmitate. This labeling, different from that of the inner membrane, could result either from transacylation activities or from a biosynthetic pathway not yet described in pea and occurring partly in the outer chloroplast envelope membrane. This metabolism would work on an oleate-rich pool of fatty acids, possibly due to the export of oleate from chloroplast toward the extrachloroplastic medium. The respective roles of each membrane for chloroplast lipid synthesis are emphasized.     

Lipid composition of outer leaflet of chloroplast outer envelope determines topology of OEP7.

OEP7, a 6.7-kDa outer envelope protein of spinach chloroplasts inserts into the outer envelope of the organelle independent of a classical cleavable targeting signal. The insertion of OEP7 was studied to describe the determinants for association with, integration into, and orientation of the protein in the outer envelope of chloroplasts. The insertion of OEP7 into the membrane is independent of outer membrane channel proteins and can be reconstituted with the use of protein-free liposomes. In situ, the binding of OEP7 to the membrane surface is not driven by electrostatic interaction because reduction of phosphatidylglycerol or phosphatidylinositol did not reduce the association with the liposomes. The positively charged amino acids flanking the transmembrane domain at the C terminus are essential to retain the native N(in)-C(out) orientation during insertion into chloroplasts. OEP7 inserts with reversed orientation into liposomes containing the average lipid composition of the outer envelopes. The native like N(in)-C(out) orientation is achieved by reduction of the phoshpatidylglycerol concentration mimicking the composition of the outer leaflet of the outer envelope of chloroplasts. We conclude that the unique lipid composition of the outer leaflet due to lipid asymmetry of the outer envelope is essential for the correct topology of OEP7.     

Envelope membranes from mature spinach chloroplasts contain a NADPH:protochlorophyllide reductase on the cytosolic side of the outer membrane

Using fluorescence spectroscopy, we have demonstrated that isolated envelope membranes from mature spinach chloroplasts catalyze the phototransformation of endogenous protochlorophyllide into chlorophyllide in presence of NADPH, but not in presence of NADH. Protochlorophyllide reductase was characterized further using monospecific antibodies (anti-protochlorophyllide reductase) raised against the purified enzyme from oat. In mature spinach chloroplasts, protochlorophyllide reductase is present only in envelope membranes. We have demonstrated that the envelope protochlorophyllide reductase, a 37,000-dalton polypeptide, is only a minor envelope component and is present on the outer surface of the outer envelope membrane. This conclusion is supported by several lines of evidence: (a) the envelope polypeptide that was immunodecorated with anti-protochlorophyllide reductase can be distinguished from the major 37,000-dalton envelope polypeptide E37 (which was identified by monospecific antibodies) only after two-dimensional polyacrylamide gel electrophoresis; (b) the envelope protochlorophyllide reductase was hydrolyzed when isolated intact chloroplasts were incubated in presence of thermolysin; and (c) isolated intact chloroplasts strongly agglutinate when incubated in presence of antibodies raised against protochlorophyllide reductase. These results demonstrate that major differences exist between chloroplasts and etioplasts with respect to protochlorophyllide reductase levels and localization. The presence on the chloroplast envelope membrane of both the substrate (protochlorophyllide) and the enzyme (protochlorophyllide reductase) necessary for chlorophyllide synthesis could have major implications for the understanding of chlorophyll biosynthesis in mature chloroplasts.     

The role of chloroplasts and microsomal fractions in polar-lipid synthesis from [1-14C]acetate by cell-free preparations from spinach (Spinacia oleracea) leaves.

1. Isolated spinach (Spinacia oleracea) chloroplasts were incapable of accumulating polar lipids when incubated with [1-14C]acetate in a cofactor-free medium. When CoA, ATP and glycerol 3-phosphate were added to incubation media, the accumulated products were non-esterified fatty acids, acyl-CoA and 1,2-diacylglycerol, all intermediates of lipid metabolism. 2. Chloroplast acyl-CoA was used to synthesize phosphatidylcholine only when a microsomal fraction was added back to the incubation medium. 3. The 1,2-diacylglycerol synthesized by isolated chloroplasts was converted almost quantitatively into diacylgalactosylglycerol when exogenous UDP-galactose was available. 4. Stereospecific analyses of the isolated lipids suggested that the diacylglycerol synthesized by isolated chloroplasts may be an important precursor for the synthesis in vivo of diacylgalactosylglycerol and phosphatidylglycerol but was unlikely to be a precursor of phosphatidylcholine. 5. A scheme for plant-lipid biosynthesis is presented that integrates the functions of chloroplasts, the cytoplasm and the endoplasmic reticulum.     

Last step in the cooperative pathway for galactolipid synthesis in spinach leaves: Formation of monogalactosyldiacylglycerol with C18 polyunsaturated acyl groups at both carbon atoms of the glycerol

In this paper, the formation of monogalactosyldiacylglycerol (MGDG) with C18 polyunsaturated acyl groups at both positions of the glycerol from phosphatidylcholine (PC) of liposomes was demonstrated by a three-step procedure: spinach chloroplasts were first incubated with liposomes of PC-containing polyunsaturated fatty acids at both positions of the glycerol in the presence of phospholipid transfer protein. Chloroplasts were then incubated with phospholipase C in order to form 1,2-diacylglycerol (1,2-DAG) from PC integrated in the chloroplast envelope. Finally, washed chloroplasts were incubated with UDP-gal and MGDG was formed by galactosylation of 1,2-DAG.     

A study on the topological distribution of phospholipids in microsomal membranes of chick brain using phospholipase C and trinitrobenzenesulfonic acid

The transbilayer distribution of phospholipids in chicken brain microsomal membranes has been investigated using trinitrobenzenesulfonic acid and phospholipase C from Clostridium weichii. The exposure of intact microsomes to trinitrobenzenesulfonic acid showed that the labelling of aminophospholipids followed biphasic kinetics, indicating that these membranes contain a fast- and a slow-reacting pool of aminophospholipids. Use of microsomes radioiodinated on their surface led to the conclusion that the fast-reacting pool may be located on the outer leaflet of the microsomal vesicles. It contains about 35% of the phosphatidylethanolamine, 29% of the ethanolamine plasmalogens and 18% of the phosphatidylserine. The treatment of intact microsomes with the phospholipase C Cl. welchii produced the hydrolysis of 50% of the phospholipids without any loss of their permeability properties, indicating that they are not permeable to the hydrolase. Phospholipids extracted from the microsomes were hydrolyzed rapidly by the phospholipase C with the exception of phosphatidylserine and phosphatidylinositol. In intact microsomes about 90% of phosphatidylcholine, 32% of ethanolamine phospholipids and 60% of sphingomyelin were accessible to the phospholipase. These results suggest that the phospholipids have an asymmetric distribution in chicken brain microsomes, the external leaflet containing about 75% of the choline phospholipids and 25% of the aminophospholipids, whereas an opposite distribution is observed in the inner leaflet.     

Preparation and characterization of membrane fractions enriched in outer and inner envelope membranes from spinach chloroplasts. I. Electrophoretic and immunochemical analyses

We have developed a fast and reliable method for the separation of two membrane fractions respectively enriched in outer and inner envelope membranes from isolated, intact, purified spinach chloroplasts kept in a hypertonic medium (0.6 M mannitol). This separation was achieved by osmotically shrinking the inner envelope membrane, thus widening the intermembrane space, and then subsequently removing the "loosened" outer envelope membrane by applying low pressure to the shrunken chloroplasts and slowly extruding them through the small aperture of a Yeda press under controlled conditions. By centrifugation of the mixture obtained through a discontinuous sucrose gradient, we were able to separate two membrane fractions having different densities (fraction 2 or light fraction, d = 1.08 g/cm3, and fraction 3 or heavy fraction, d = 1.13 g/cm3). The recent characterization of polypeptides localized on the outer envelope membrane from spinach chloroplasts, E10 and E24 (Joyard, J., Billecocq, A., Bartlett, S. G., Block, M. A., Chua, N.-H., and Douce, R. J. Biol. Chem., 258, 10000-10006) enabled us to characterize our two membrane fractions. Analyses of the polypeptides by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis and immunoblotting have shown that fraction 2 (light fraction) was completely devoid of polypeptide E30, which is involved in the transport of phosphate across the inner envelope membrane, but was enriched in polypeptides E10 and E24. The reverse was true for fraction 3 (heavy fraction). Under these conditions, it is clear that fraction 2 is strongly enriched in outer envelope membrane whereas fraction 3 consisted mostly of inner envelope membrane. Indeed, by immunoelectrophoresis, we were able to demonstrate that, on a protein basis, fraction 2 contained about 90% of outer membrane, whereas fraction 3 contained about 80% of inner membrane. Further characterization of the outer envelope membrane was achieved by using thermolysin, a nonpenetrant protease.     

Asymmetry of the phospholipid bilayer of rat liver endoplasmic reticulum

The phospholipids of intact microsomal membranes were hydrolysed 50% by phospholipase C of Clostridium welchii, without loss of the secretory protein contents of the vesicle, which are therefore not permeable to the phospholipase. Phospholipids extracted from microsomes and dispersed by sonication were hydrolysed rapidly by phospholipase C-Cl. welchii with the exception of phosphatidylinositol. Assuming that only the phospholipids of the outside of the bilayer of the microsomal membrane are hydrolysed in intact vesicles, the composition of this leaflet was calculated as 84% phosphatidylcholine, 8% phosphatidylethanolamine, 9% sphingomyelin and 4% phosphatidylserine, and that of the inner leaflet 28% phosphatidylcholine, 37% phosphatidylethanolamine, 6% phosphatidylserine and 5% sphingomyelin. Microsomal vesicles were opened and their contents released in part by incubation with deoxycholate (0.098%) lysophosphatidylcholine (0.005%) or treatment with the French pressure cell. Under these conditions, hydrolysis of the phospholipids by phospholipase C-Cl. welchii was increased and this was mainly due to increased hydrolysis of those phospholipids assigned to the inner leaflet of the bilayer, phosphatidylethanolamine and phosphatidylserine. Phospholipase A₂ of bee venom and phospholipase C of Bacillus cereus caused rapid loss of vesicle contents and complete hydrolysis of the membrane phospholipids, with the exception of sphingomyelin which is not hydrolysed by the former enzyme.     

Sulfolipid is a potential candidate for annexin binding to the outer surface of chloroplast

Using a subcellular-specific proteomic approach, we have identified by protein microsequencing, a putative 35-kDa annexin from among the chloroplast envelope polypeptides. To confirm this identification, we demonstrate that (a) a 35-kDa protein, identified as annexin by antibody cross-reactivity, co-purifies with Percoll-purified chloroplasts and their envelope membranes when extracted in the presence of Ca(2+) and (b) the native spinach annexin protein binds to chloroplast-specific lipids in a Ca(2+)-dependent manner. The binding of the spinach annexin to these glycerolipids occurs at similar Ca(2+) concentrations as those, which promote the interaction of annexins to phospholipids in other membranes. Among chloroplast glycerolipids known to be accessible on the cytosolic face (outer leaflet) of the outer envelope membrane, sulfolipid, and probably phosphatidylinositol, would be the sole candidates for a putative Ca(2+)-dependent interaction of annexin with the chloroplast surface.     

Phospholipid metabolism in plant mitochondria: submitochondrial sites of synthesis

Intact mitochondria from the endosperm of castor bean were isolated on linear sucrose gradients. These mitochondria were ruptured and the membranes separated on discontinuous sucrose gradients into outer membrane, intact inner membrane, and ruptured inner membrane fractions. Each membrane fraction was examined for its capacity to synthesize phosphatidylglycerol, CDP-diglyceride, phosphatidylcholine via methylation, and phosphatidic acid. The syntheses of phosphatidylglycerol, CDP-diglyceride, and phosphatidylcholine were localized exclusively in the inner mitochondrial membrane fractions while phosphatidic acid synthesis occurred in both the inner and outer mitochondrial membranes.     

Asymmetry of lipid organization in cholinergic synaptic vesicle membranes.

The lipid composition of purified Torpedo cholinergic synaptic vesicles was determined and their distribution between the inner and outer leaflets of the vesicular membrane was investigated. The vesicles contain cholesterol and phospholipids at a molar ratio of 0.63. The vesicular phospholipids are (mol% of total phospholipids): phosphatidylcholine (40.9); phosphatidylethanolamine (24.6); plasmenylethanolamine (11.5); sphingomyelin (12); phosphatidylserine (7.3); phosphatidylinositol (3.7). The asymmetry of the synaptic vesicle membranes was investigated by two independent approaches: (a) determining accessibility of the amino lipids to the chemical label trinitrobenzenesulphonic acid (TNBS); (b) determining accessibility of the vesicular glycerophospholipids to phospholipase C (Bacillus cereus). TNBS was found to render the vesicles leaky and thus cannot be used reliably to determine the asymmetry of Torpedo synaptic vesicle membranes. Incubation of the vesicles with phospholipase C (Bacillus cereus) results in biphasic hydrolysis of the vesicular glycerophospholipids. About 45% of the phospholipids are hydrolysed in less than 1 min, during which no vesicular acetylcholine is released. In the second phase, the hydrolysis of the phospholipids slows down markedly and is accompanied by loss of all the vesicular acetylcholine. These findings suggest that the lipids hydrolysed during the first phase are those comprising the outer leaflet. Analysis of the results thus obtained indicate that the vesicular membrane is asymmetric: all the phosphatidylinositol, 77% of the phosphatidylethanolamine, 47% of the plasmenylethanolamine and 58% of the phosphatidylcholine were found to reside in the outer leaflet. Since phosphatidylserine is a poor substrate for phospholipase C (B. cereus), its distribution between the two leaflets of the synaptic vesicle membrane is only suggestive.     

Phosphatidylglycerol synthesis in pea chloroplasts: pathway and localization

Isolated intact pea chloroplasts synthesized phosphatidylglycerol from either [¹⁴C]acetate or [¹⁴C]glycerol 3-phosphate. Both time-course and pulse-chase labeling studies demonstrated a precursor-product relationship between newly synthesized phosphatidic acid and newly synthesized phosphatidylglycerol.

The synthesis both of CDP-diacylglycerol from exogenous phosphatidic acid and CTP, and of phosphatidylglycerol from exogenous CDP-diacylglycerol and glycerol 3-phosphate, could be assayed in fractions obtained from disrupted chloroplasts. Moreover, the enzymes catalyzing these reactions were localized in the inner envelope membrane. Exogenous phosphatidic acid was incorporated into phosphatidylglycerol, but only following its incorporation into CDP-diacylglycerol. Finally, radio-active phosphatidic acid synthesized in the envelope membranes from [¹⁴C]palmitoyl-ACP and 1-oleoyl-glycerol 3-phosphate was sequentially incorporated into labeled CDP-diacylglycerol and phosphatidylglycerol upon the addition of appropriate substrates and cofactors. Thus, we have demonstrated that (a) the synthesis of phosphatidylglycerol in chloroplasts occurs by the pathway: phosphatidic acid → CDP-diacylglycerol →→ phosphatidylglycerol, and (b) phosphatidylglycerol synthesis is located in the inner envelope membrane.

     

Asymmetric synthesis followed by transmembrane movement of phosphatidylethanolamine in rat liver endoplasmic reticulum

Studies with phospholipase C have indicated that two-thirds of the phosphatidylethanolamine of rat liver endoplasmic reticulum is located in the inner leaflet of the membrane bilayer. Phosphatidyl[¹⁴C]ethanolamine is synthesised in microsomes incubated with CDP[¹⁴C]ethanolamine. Using phospholipase C as a probe we have observed that the labelled phospholipid is initially (1–2 min) concentrated in the ‘outer leaflet’ of the membrane bilayer. The specific activity of this pool of phosphatidylethanolamine was 3.5 times that of the inner leaflet. If, however, the microsomes were opened with 0.4% taurocholate or the French pressure cell to make both sides of the bilayer available to phospholipase C, the phosphatidylethanolamine behaves as a single pool for hydrolysis. On longer incubation, up to 30 min, with CDP[¹⁴C]ethanolamine the specific activity of the outer leaflet phosphatidylethanolamine becomes close to that of the inner leaflet. In chase experiments, in which microsomal phosphatidylethanolamine was labelled by incubation with CDP[¹⁴C]ethanolamine for 1 min, the reaction stopped by addition of calcium, and the microsomes isolated by centrifugation and reincubated, labelled phosphatidylethanolamine was transferred from the ‘outer leaflet’ to the ‘inner leaflet’, so that both were equally labelled. These observations suggest that phosphatidylethanolamine is synthesised at the cytoplasmic leaflet of the endoplasmic reticulum and subsequently transferred across the membrane to the cisternal leaflet of the bilayer. Transmembrane movement is apparently temperature-dependent and independent of continued synthesis of phosphatidylethanolamine.     

The involvement of cytosolic lipases in converting phosphatidyl choline to substrate for galactolipid synthesis in the chloroplast envelope

Here we report that cytosolic phospholipases are involved in the utilization of phosphatidylcholine (PC) as substrate for chloroplast-localized synthesis of monogalactosyldiacylglycerol (MGDG). Isolated chloroplasts were pre-incubated with lysoPC and [14C]18:0-CoA to form [14C]PC. When soluble plant proteins (cytosol) and UDP-galactose were added, [14C] MGDG was formed. An inhibitor of phospholipase D markedly lowered the formation of [14C]MGDG, whereas thermolysin pretreatment of the chloroplasts was without effect. The cytosolic activity resided in the >100-kDa fraction. In a second approach, [14C]PC-containing lipid mixtures were incubated with cytosol. Degradation of [14C]PC to [14C]diacylglycerol was highest when the lipid composition of the mixture mimicked that of the outer chloroplast envelope. We also investigated whether PC of extraplastidic origin could function as substrate for MGDG synthesis. Isolated chloroplasts were incubated with enriched endoplasmic reticulum containing radiolabelled acyl lipids. In the presence of cytosol and UDP-galactose, there was a time-dependent transfer of [14C]PC from this fraction to chloroplasts, where [14C]MGDG was formed. We conclude that chloroplasts recruit cytosolic phospholipase D and phosphatidic acid phosphatase to convert PC to diacylglycerol. Apparently, these lipases do not interact with chloroplast surface proteins, but rather with outer membrane lipids, either for association to the envelope or for substrate presentation.     

Targeting of proteins to the outer envelope membrane uses a different pathway than transport into chloroplasts.

The chloroplastic envelope is composed of two membranes, inner and outer, each with a distinct set of polypeptides. Like proteins in other chloroplastic compartments, most envelope proteins are synthesized in the cytosol and post-translationally imported into chloroplasts. Considerable knowledge has been obtained concerning protein import proteins. We isolated a cDNA clone from pea that encodes a 14-kilodalton outer envelope membrane protein. The precursor form of this protein does not possess a cleavable transit peptide and its import into isolated chloroplasts does not require either ATP or a thermolysin-sensitive component on the chloroplastic surface. These findings, together with similar observations made with a spinach chloroplastic outer membrane protein, led us to propose that proteins destined for the outer membrane of the chloroplastic envelope follow an import pathway distinct from that followed by proteins destined for other chloroplastic compartments.     

Topological Biosynthesis of Phosphatidylcholine in Brain Microsomes

The sidedness of the biosynthesis of phosphatidylcholine and its transbilayer movement in brain microsomes were investigated. Microsomes were labelled in vitro or in vivo either through Kennedy's pathway or by the base-exchange reaction. The vesicles were treated with phospholipase C under conditions where only the phospholipids present in the external leaflet were hydrolyzed. The incubation of microsomes with CDP-[14C]choline or [14C]choline showed that most of the newly synthesized phosphatidylcholine molecules were localized in the external leaflet. With time a few molecules were transferred into the inner leaflet. When phosphatidylcholine was labelled in vivo by intraventricular injection of [3H]choline the specific activities of the phosphatidylcholine in the outer leaflet were higher than those in the inner leaflet after short times of labelling but became similar after long times of labelling. The results suggest that in brain microsomes the synthesis of phosphatidylcholine through Kennedy's pathway or by the base-exchange reaction takes place on the external leaflet which corresponds to the cytoplasmic one in situ. The transfer of these molecules from the outer leaflet to the inner one is a slow process and the mechanisms that control the transbilayer movement of the phosphatidylcholine seem to be independent of those that control their biosynthesis.     

Assay of Proton-Coupled Glycolate and D-Glycerate Transport into Chloroplast Inner Envelope Membrane Vesicles by Stopped-Flow Fluorescence

The transport of glycolate and D-glycerate across the inner envelope membrane of intact chloroplasts is rapid and mediated by a translocator with proton/substrate symport activity. The true initial rate of glycolate or D-glycerate transport could not be measured by conventional methods. To resolve the initial rates of glycolate and D-glycerate transport, a stopped-flow fluorescence assay was developed that allows the indirect observation of transport from about 4 ms after mixing. Inner envelope vesicles from pea (Pisum sativum) or spinach (Spinacia oleracea) chloroplasts were loaded with the fluorescent pH indicator pyranine (8-hydroxypyrene-1,3,6-trisulfonic acid) by a freeze-thaw sonication protocol. A rapid quenching of pyranine fluorescence was detected after mixing the vesicles with either glycolate or D-glycerate. This quenching was the result of acidification of the interior of the vesicles. D-Glycerate- or glycolate-induced acidification displayed saturation kinetics and was inhibited by pretreatment of the vesicles with N-ethylmaleimide. D-Glycerate was more effective than L-glycerate in causing the pH decrease. Also, L-mandelate inhibited D-glycerate-induced acidification much more strongly than D-mandelate. The glycolate/D-glycerate-induced pH decrease is consistent with glycolate/D-glycerate translocator activity. The assay was placed on a quantitative basis by converting fluorescence changes to pH and measuring the internal buffering capacity of the vesicles. The rates of transport across the inner envelope membrane were estimated to be as fast, if not faster, than those of transport in intact chloroplasts.