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.     

Biosynthesis of digalactosyldiacylglycerol in plastids from 16:3 and 18:3 plants

Intact chloroplasts isolated from leaves of eight species of 16:3 and 18:3 plants and chromoplasts isolated from Narcissus pseudonarcissus L. flowers synthesize galactose-labeled mono-, di-, and trigalactosyldiacylglycerol (MGDG, DGDG, and TGDG) when incubated with UDP-[6-³H]galactose. In all plastids, galactolipid synthesis, and especially synthesis of DGDG and TGDG, is reduced by treatment of the organelles with the nonpenetrating protease thermolysin. Envelope membranes isolated from thermolysin-treated chloroplasts of Spinacia oleracea L. (16:3 plant) and Pisum sativum L. (18:3 plant) or membranes isolated from thermolysin-treated chromoplasts are strongly reduced in galactolipid:galactolipid galactosyltransferase activity, but not with regard to UDP-Gal:diacylglycerol galactosyltransferase. For the intact plastids, this indicates that thermolysin treatment specifically blocks DGDG (and TGDG) synthesis, whereas MGDG synthesis is not affected. Neither in chloroplast nor in chromoplast membranes is DGDG synthesis stimulated by UDP-Gal. DGDG synthesis in S. oleracea chloroplasts is not stimulated by nucleoside 5′-diphospho digalactosides. Therefore, galactolipid:galactolipid galactosyltransferase is so far the only detectable enzyme synthesizing DGDG. These results conclusively suggest that the latter enzyme is located in the outer envelope membrane of different types of plastids and has a general function in DGDG synthesis, both in 16:3 and 18:3 plants.     

Acyl-CoA Synthetase Is Located in the Outer Membrane and Acyl-CoA Thioesterase in the Inner Membrane of Pea Chloroplast Envelopes

Both acyl-CoA synthetase and acyl-CoA thioesterase activities are present in chloroplast envelope membranes. The functions of these enzymes in lipid metabolism remains unresolved, although the synthetase has been proposed to be involved in either plastid galactolipid synthesis or the export of plastid-synthesized fatty acids to the cytoplasm. We have examined the locations of both enzymes within the two envelope membranes of pea (Pisum sativum var Laxton's Progress No. 9) chloroplasts. Inner and outer envelope membranes were purified from unfractionated envelope preparations by linear density sucrose gradient centrifugation. Acyl-CoA synthetase was located in the outer envelope membrane while acyl-CoA thioesterase was located in the inner envelope membrane. Thus, it seems unlikely that the synthetase is directly involved in galactolipid assembly. Instead, its localization supports the hypothesis that it functions in the transport of plastid-synthesized fatty acids to the endoplasmic reticulum.     

Feedback inhibition of phosphatidate phosphatase from spinach chloroplast envelope membranes by diacylglycerol.

Because the envelope phosphatidate phosphatase plays a pivotal role in chloroplast glycerolipid metabolism, we have analyzed whether diacylglycerol could be a regulatory factor of the enzyme. Using isolated envelope membranes in which the level of diacylglycerol was modified by thermolysin treatment of intact chloroplasts to destroy the galactolipid:galactolipid galactosyltransferase, we have demonstrated that phosphatidate phosphatase activity was reduced when the membrane was enriched in diacylglycerol. All 1,2-diacylglycerol molecular species assayed were demonstrated to inhibit the enzyme to about the same extent. Kinetic studies with envelope from thermolysin-treated chloroplasts were performed in the absence and presence of diacylglycerol, and diacylglycerol was shown to be a powerful competitive inhibitor of the reaction. Finally, using isolated intact spinach chloroplasts, we have demonstrated that in situ phosphatidate phosphatase activity can be modulated by the level of diacylglycerol present in the membrane. The relevance of phosphatidate phosphatase inhibition by diacylglycerol in the regulation of chloroplast glycerolipid biosynthesis is discussed.     

Synthesis of mono- and digalactosyldiacylglycerol in isolated spinach chloroplasts

Purified, intact chloroplasts of Spinacia oleracea L. synthesize galactose-labeled mono- and digalactosyldiacylglycerol (MGDG and DGDG) from UDP-[U-¹⁴C]galactose. In the presence of high concentrations of unchelated divalent cations they also synthesize tri- and tetra-galactosyldiacylglycerol. The acyl chains of galactose-labeled MGDG are strongly desaturated and such MGDG is a good precursor for DGDG and higher oligogalactolipids. The synthesis of MGDG is catalyzed by UDP-Gal:sn-1,2-diacylglycerol galactosyltransferase, and synthesis of DGDG and the oligogalactolipids is exclusively catalyzed by galactolipid:galactolipid galactosyltransferase. The content of diacylglycerol in chloroplasts remains low during UDP-Gal incorporation. This indicates that formation of diacylglycerol by galactolipid:galactolipid galactosyltransferase is balanced with diacylglycerol consumption by UDP-Gal:diacylglycerol galactosyltransferase for MGDG synthesis. Incubation of intact spinach chloroplasts with [2-¹⁴C]acetate or sn-[U-¹⁴C]glycerol-3-P in the presence of Mg²⁺ and unlabeled UDP-Gal resulted in high ¹⁴C incorporation into MGDG, while DGDG labeling was low. This de novo made MGDG is mainly oligoene. Its conversion into DGDG is also catalyzed, at least in part, by galactolipid:galactolipid galactosyltransferase.     

A Close Relationship between Increases in Galactosyltransferase Activity and the Accumulation of Galactolipids during Plastid Development in Cucumber Seedlings

Changes in the activity of UDP-galactose:diacylglycerol galactosyltransferase(UDGT), a key enzyme in galactolipid biosynthesis, during germinationwere investigated in cucumber (Cucumis sativus L. cv. Aonagajibai)seedlings. After germination, UDGT activity increased duringgrowth in darkness for 4 days, reaching 10 times the activityin ungerminated seeds. Illumination of 4-day-old dark-grownseedlings strongly stimulated the activity. By contrast, inseedlings grown continuously in darkness, the increase in UDGTactivity ceased after 4 days and the activity remained constantthereafter. A similar increase in the specific activity of UDGTwas observed i n the envelope fraction from seedlings, indicatingthat the increase in the enzymatic activity preceded synthesisof other proteins in the envelope membrane. Coincident withthe change in the enzymatic activity, here was an increase inlevels of monogalactosyl diacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), two major constituents of chloroplastmembrane lipids, in the germinated seedlings. Cycloheximideinhibited the light-mediated increase in the enzymatic activityby illumination of 4-day-old dark-grown seedlings, and, as aconsequence, it inhibited the accumulation of MGDG and DGDG.It was clear, therefore, that protein synthesis was necessaryduring this activation. Addition of a cytokinin, benzyladenine(BA), stimulated the increase in the UDGT activity. The increasein the UDGT activity caused by BA was accompanied by the accumulationof galactolipids, as in the case of the activation by light.These results suggest that activation of the final reactionin the synthesis of MGDG, which is catalyzed by the galactosyl-transferase,contributes to the accumulation of galactolipids during thedevelopment of the chloroplast membrane. (Received December 3, 1994; Accepted July 3, 1995)     

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 synthesis and metabolism in the plastid envelope

Plastid envelope membranes play a major role in the biosynthesis of glycerolipids. In addition, plastids are characterized by the occurrence of plastid-specific membrane glycolipids (galactolipids, a sulfolipid). Plant lipid metabolism therefore has unique features, when compared to that of other eukaryotic organisms, such as animals and yeast. However, the glycerolipid biosynthetic pathway in chloroplasts is almost identical to that found in cyanobacteria, and reflects the prokaryotic origin of the chloroplast. Fatty acids generated in the plastid stroma are substrates for a whole set of enzymes involved in the synthesis of polar lipids of plastid membranes such as galactolipids, the sulfolipid, the phosphatidylglycerol. In addition, fatty acids are exported outside the plastid where they are used for extraplastidial polar lipid synthesis (phosphatidylcholine, phosphatidylethanolamine, etc.). Various desaturation steps leading to the formation of polyunsaturated fatty acids occur in various cell compartments, especially in chloroplasts, using fatty acids esterified to polar lipids as substrates. Furthermore, plant glycerolipids can be metabolized by a series of very active envelope enzymes, such as the galactolipid:galactolipid galactosyltransferase and the acyl-galactolipid forming enzyme. The physiological significance of these enzymes is however largely unknown. One of the most active pathways involved in lipid metabolism and present in envelope membranes is the oxylipin pathway: polyunsaturated fatty acids that are released from polar lipids under various conditions (injury, pathogen attack) are converted to oxylipin. Thus, the plastid envelope membranes are also involved in the formation of signalling molecules.     

Preparation and characterization of envelope membranes from nongreen plastids

We have developed a reliable procedure for the purification of envelope membranes from cauliflower (Brassica oleracea L.) bud plastids and sycamore (Acer pseudoplatanus L.) cell amyloplasts. After disruption of purified intact plastids, separation of envelope membranes was achieved by centrifugation on a linear sucrose gradient. A membrane fraction, having a density of 1.122 grams per cubic centimeter and containing carotenoids, was identified as the plastid envelope by the presence of monogalactosyldiacylglycerol synthase. Using antibodies raised against spinach chloroplast envelope polypeptides E24 and E30, we have demonstrated that both the outer and the inner envelope membranes were present in this envelope fraction. The major polypeptide in the envelope fractions from sycamore and cauliflower plastids was identified immunologically as the phosphate translocator. In the envelope membranes from cauliflower and sycamore plastids, the major glycerolipids were monogalactosyldiacylglycerol, digalactosyldiacylglycerol, and phosphatidylcholine. Purified envelope membranes from cauliflower bud plastids and sycamore amyloplasts also contained a galactolipid:galactolipid galactosyltransferase, enzymes for phosphatidic acid and diacylglycerol biosynthesis, acyl-coenzyme A thioesterase, and acyl-coenzyme A synthetase. These results demonstrate that envelope membranes from nongreen plastids present a high level of homology with chloroplasts envelope membranes.     

Final step of phosphatidic Acid synthesis in pea chloroplasts occurs in the inner envelope membrane

The second enzyme of phosphatidic acid synthesis from glycerol-3-phosphate, 1-acylglycerophospate acyltransferase, was localized to the inner envelope membrane of pea chloroplasts. The activity of this enzyme was measured by both a coupled enzyme assay and a direct enzyme assay. Using the coupled enzyme assay, phosphatidic acid phosphatase was also localized to the inner envelope membrane, although this enzyme has very low activity in pea chloroplasts. The addition of UDP-galactose to unfractionated pea chloroplast envelope preparations did not result in significant conversion of newly synthesized diacylglycerol to monogalactosyldiacylglycerol. Thus, the envelope synthesized phosphatidic acid may not be involved in galactolipid synthesis in pea chloroplasts.     

Structural insights and membrane binding properties of MGD1, the major galactolipid synthase in plants

Monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are the major lipid components of photosynthetic membranes, and hence the most abundant lipids in the biosphere. They are essential for assembly and function of the photosynthetic apparatus. In Arabidopsis, the first step of galactolipid synthesis is catalyzed by MGDG synthase 1 (MGD1), which transfers a galactosyl residue from UDP‐galactose to diacylglycerol (DAG). MGD1 is a monotopic protein that is embedded in the inner envelope membrane of chloroplasts. Once produced, MGDG is transferred to the outer envelope membrane, where DGDG synthesis occurs, and to thylakoids. Here we present two crystal structures of MGD1: one unliganded and one complexed with UDP. MGD1 has a long and flexible region (approximately 50 amino acids) that is required for DAG binding. The structures reveal critical features of the MGD1 catalytic mechanism and its membrane binding mode, tested on biomimetic Langmuir monolayers, giving insights into chloroplast membrane biogenesis. The structural plasticity of MGD1, ensuring very rapid capture and utilization of DAG, and its interaction with anionic lipids, possibly driving the construction of lipoproteic clusters, are consistent with the role of this enzyme, not only in expansion of the inner envelope membrane, but also in supplying MGDG to the outer envelope and nascent thylakoid membranes.     

Tic20 and Tic22 Are New Components of the Protein Import Apparatus at the Chloroplast Inner Envelope Membrane

Two components of the chloroplast envelope, Tic20 and Tic22, were previously identified as candidates for components of the general protein import machinery by their ability to covalently cross-link to nuclear-encoded preproteins trapped at an intermediate stage in import across the envelope (Kouranov, A., and D.J. Schnell. 1997. J. Cell Biol. 139:1677–1685). We have determined the primary structures of Tic20 and Tic22 and investigated their localization and association within the chloroplast envelope. Tic20 is a 20-kD integral membrane component of the inner envelope membrane. In contrast, Tic22 is a 22-kD protein that is located in the intermembrane space between the outer and inner envelope membranes and is peripherally associated with the outer face of the inner membrane. Tic20, Tic22, and a third inner membrane import component, Tic110, associate with import components of the outer envelope membrane. Preprotein import intermediates quantitatively associate with this outer/inner membrane supercomplex, providing evidence that the complex corresponds to envelope contact sites that mediate direct transport of preproteins from the cytoplasm to the stromal compartment. On the basis of these results, we propose that Tic20 and Tic22 are core components of the protein translocon of the inner envelope membrane of chloroplasts.     

Uridine-diphospho-sulfoquinovose: diacylglycerol sulfoquinovosyltransferase activity is concentrated in the inner membrane of chloroplast envelopes

In experiments on the assembly of the sulfolipid sulfoquinovosyl diacylglycerol in envelope membranes of chloroplasts, UDP-sulfoquinovose (UDPS) was used with highest efficiency, and the corresponding enzyme, UDP-sulfoquinovose:diacylglycerol sulfoquinovosyltransferase, was partially characterized (E. Heinz et al., 1989, Eur J Biochem 184: 445–453). Here, we identified ³⁵S- and ³³P-labelled UDPS from various photosynthetically active organisms, suggesting that the sulfosugar nucleotide used for sulfolipid biosynthesis throughout the plant kingdom, including phototrophic bacteria, may indeed be UDPS. For attribution of the sulfolipid synthase to one of the two plastidial envelope membranes, these membranes were isolated from pea and spinach chloroplasts. The sulfoquinovosyltransferase was localized in the inner membrane of envelopes, which also contains the competing UDP-galactose:diacylglycerol galactosyltransferase. In contrast to the sulfoquinovosyltransferase, a substantial proportion of the galactosyltransferase was found in the outer membranes of envelopes from pea chloroplasts. Received: 6 October 1997 / Accepted: 31 January 1998     

Galactolipid Synthesis in Vicia faba Leaves: III. Site(s) of Galactosyl Transferase Activity

Leaves of Vicia faba were fed ¹⁴CO₂ in light for periods of up to 6 hours. At intervals, leaf samples were homogenized and separated into fractions which contained “broken” and “intact” chloroplasts, and three other high speed centrifugal fractions containing other cell membranes and chloroplast envelopes. Analyses of the radioactive labeling of galactose from the galactolipids in these fractions and in purified chloroplast envelopes indicated that the major site of galactosyl transferase enzyme activity was in the chloroplast envelope. The data suggest that in time much of the radioactive galactolipid was transferred from the envelope to the thylakoid-containing fractions. The major site of galactolipid synthesis appears to be in the envelope but there is some evidence of another site in the thylakoids.     

Lipid trafficking between the endoplasmic reticulum and the plastid in Arabidopsis requires the extraplastidic TGD4 protein

The development of chloroplasts in Arabidopsis thaliana requires extensive lipid trafficking between the endoplasmic reticulum (ER) and the plastid. The biosynthetic enzymes for the final steps of chloroplast lipid assembly are associated with the plastid envelope membranes. For example, during biosynthesis of the galactoglycerolipids predominant in photosynthetic membranes, galactosyltransferases associated with these membranes transfer galactosyl residues from UDP-Gal to diacylglycerol. In Arabidopsis, diacylglycerol can be derived from the ER or the plastid. Here, we describe a mutant of Arabidopsis, trigalactosyldiacylglycerol4 (tgd4), in which ER-derived diacylglycerol is not available for galactoglycerolipid biosynthesis. This mutant accumulates diagnostic oligogalactoglycerolipids, hence its name, and triacylglycerol in its tissues. The TGD4 gene encodes a protein that appears to be associated with the ER membranes. Mutant ER microsomes show a decreased transfer of lipids to isolated plastids consistent with in vivo labeling data, indicating a disruption of ER-to-plastid lipid transfer. The complex lipid phenotype of the mutant is similar to that of the tgd1,2,3 mutants disrupted in components of a lipid transporter of the inner plastid envelope membrane. However, unlike the TGD1,2,3 complex, which is proposed to transfer phosphatidic acid through the inner envelope membrane, TGD4 appears to be part of the machinery mediating lipid transfer between the ER and the outer plastid envelope membrane. The extent of direct ER-to-plastid envelope contact sites is not altered in the tgd4 mutant. However, this does not preclude a possible function of TGD4 in those contact sites as a conduit for lipid transfer between the ER and the plastid.     

Intraplastidial lipid trafficking: Regulation of galactolipid release from isolated chloroplast envelope

A method is described for cell-free studies of lipid release from isolated chloroplast envelope. The isolated membrane fraction incorporated radiolabeled galactose into galactolipids, predominantly monogalactosyldiacylglycerol, prior to immobilization of the membrane vesicles onto strips of nitrocellulose. The strips with immobilized membrane were individually incubated with various co-factors and the incubations were terminated by removing the strips. Radioactivity was determined for the strips with immobilized membrane as well as for the material released during the assay. The release of galactolipids from immobilized chloroplast envelope was time- and temperature dependent, required stroma protein(s) and was further stimulated by hydrolysable ATP, GTP and ≤50 μ M acyl-CoAs, of which 16:1-CoA was the most stimulative. To investigate whether guanine nucleotide-binding proteins could be involved, stroma and envelope were independently or together incubated with [ α -³²P]GTP or [ Γ -³²P]GTP. Stroma and envelope proteins were phosphorylated and the envelope fraction contained GMP/GDP binding proteins as well. When the fractions were co-incubated, the patterns of protein phosphorylation and guanine nucleotide binding was different compared to the additive effects of the separate fractions, suggesting that guanine nucleotides may have roles in galactolipid release in addition to providing energy. The results point to several similarities between the regulation of galactolipid release from isolated chloroplast envelope and the regulation of vesicular trafficking among animal and yeast cytosolic membranes, although other mechanisms for lipid release cannot, at this stage, be ruled out.     

Isolation and characterization of an Arabidopsis mutant deficient in the thylakoid lipid digalactosyl diacylglycerol.

The galactolipids monogalactosyl and digalactosyl diacylglycerol occur in all higher plants and are the predominant lipid components of chloroplast membranes. They are thought to be of major importance to chloroplast morphology and physiology, although direct experimental evidence is still lacking. The enzymes responsible for final assembly of galactolipids are associated with the envelope membranes of plastids, and their biochemical analysis has been notoriously difficult. Therefore, we have chosen a genetic approach to study the biosynthesis and function of galactolipids in higher plants. We isolated a mutant of Arabidopsis that is deficient in digalactosyl diacylglycerol by directly screening a mutagenized M2 population for individuals with altered leaf lipid composition. This mutant carries a recessive nuclear mutation at a single locus designated dgd1. Backcrossed mutants show stunted growth, pale green leaf color, reduced photosynthetic capability, and altered thylakoid membrane ultrastructure.     

Three enzyme systems for galactoglycerolipid biosynthesis are coordinately regulated in plants

Galactoglycerolipids, in which galactose is bound at the glycerol sn-3 position in O-glycosidic linkage to diacylglycerol, are abundant in plants and photosynthetic bacteria, where they constitute the bulk of the polar lipids of the photosynthetic membranes. Galactoglycerolipid biosynthesis in plants is highly compartmentalized involving enzymes at the endoplasmic reticulum and the two chloroplast envelopes. This peculiar organization requires extensive trafficking of lipid precursors. It is now increasingly apparent that there are three different sets of lipid galactosyltransferases capable of galactoglycerolipid biosynthesis in the model plant Arabidopsis. Two enzymes, MGD1 and DGD1, provide the bulk of galactoglycerolipids in the chloroplast and in photosynthetic tissues in general. Under phosphate-limited growth conditions and in non-photosynthetic tissues MGD2/3 and DGD2 are highly active. Moreover, galactoglycerolipids produced by this second pathway are often found in extraplastidic membranes. Although these galactosyltransferases use UDP-Gal as the galactose donor, a third pathway involves a processive enzyme, which transfers galactose from one galactolipid to another.     

Localization and synthesis of prenylquinones in isolated outer and inner envelope membranes from spinach chloroplasts

The prenylquinone content and biosynthetic capabilities of membrane fractions enriched in outer and inner envelope membranes from spinach chloroplasts were analyzed. Both envelope membranes contain prenylquinones, and in almost similar amounts (on a protein basis). However, the outer envelope membrane contains more alpha-tocopherol than the inner one although this prenylquinone is the major one in both fractions. On the contrary, plastoquinone-9 is present in higher amounts in the inner envelope membrane than in the outer one. In addition, it has been demonstrated that all the enzymes involved in the last steps of alpha-tocopherol and plastoquinone-9 biosynthesis, i.e., homogentisate decarboxylase polyprenyltransferase, S-adenosyl-methionine:methyl-6-phytylquinol methyltransferase, S-adenosyl-methionine: alpha-tocopherol methyltransferase, homogentisate decarboxylase solanesyltransferase, S-adenosyl-methionine:methyl-6-solanesylquinol methyltransferase, and possibly 2,3-dimethylphytylquinol cyclase, are localized on the inner envelope membrane. These results demonstrate that the inner membrane of the chloroplast envelope plays a key role in chloroplast biogenesis, and especially for the synthesis of the two major plastid prenylquinones.     

Green light for galactolipid trafficking

Galactolipids not only play a crucial role in photosynthesis but are also important for the adaptation of membrane-lipid composition in plants to phosphate-limiting conditions. The enzymes of galactolipid assembly have been localised to the envelope membranes of chloroplasts. Lipid trafficking is essential for galactolipid synthesis and redistribution because lipid precursors originate from two compartments, the endoplasmic reticulum (ER) and the plastid, and because galactolipids have to be transported to extraplastidial membranes during phosphate deprivation. Analysis of Arabidopsis mutants that are impaired in galactolipid synthesis (i.e. dgd1 and dgd2) or in ER-to-plastid lipid transport (i.e. tgd1) has resulted in the identification of a processive galactosyltransferase whose function is still enigmatic.