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.     

The Phosphatidic Acid Binding Site of the Arabidopsis Trigalactosyldiacylglycerol 4 (TGD4) Protein Required for Lipid Import into Chloroplasts

Chloroplast membrane lipid synthesis relies on the import of glycerolipids from the ER. The TGD (TriGalactosylDiacylglycerol) proteins are required for this lipid transfer process. The TGD1, -2, and -3 proteins form a putative ABC (ATP-binding cassette) transporter transporting ER-derived lipids through the inner envelope membrane of the chloroplast, while TGD4 binds phosphatidic acid (PtdOH) and resides in the outer chloroplast envelope. We identified two sequences in TGD4, amino acids 1–80 and 110–145, which are necessary and sufficient for PtdOH binding. Deletion of both sequences abolished PtdOH binding activity. We also found that TGD4 from 18:3 plants bound specifically and with increased affinity PtdOH. TGD4 did not interact with other proteins and formed a homodimer both in vitro and in vivo. Our results suggest that TGD4 is an integral dimeric β-barrel lipid transfer protein that binds PtdOH with its N terminus and contains dimerization domains at its C terminus.     

TGD4 involved in endoplasmic reticulum-to-chloroplast lipid trafficking is a phosphatidic acid binding protein

The synthesis of galactoglycerolipids, which are prevalent in photosynthetic membranes, involves enzymes at the endoplasmic reticulum (ER) and the chloroplast envelope membranes. Genetic analysis of trigalactosyldiacylglycerol (TGD) proteins in Arabidopsis has demonstrated their role in polar lipid transfer from the ER to the chloroplast. The TGD1, 2, and 3 proteins resemble components of a bacterial-type ATP-binding cassette (ABC) transporter, with TGD1 representing the permease, TGD2 the substrate binding protein, and TGD3 the ATPase. However, the function of the TGD4 protein in this process is less clear and its location in plant cells remains to be firmly determined. The predicted C-terminal β-barrel structure of TGD4 is weakly similar to proteins of the outer cell membrane of Gram-negative bacteria. Here, we show that, like TGD2, the TGD4 protein when fused to DsRED specifically binds phosphatidic acid (PtdOH). As previously shown for tgd1 mutants, tgd4 mutants have elevated PtdOH content, probably in extraplastidic membranes. Using highly purified and specific antibodies to probe different cell fractions, we demonstrated that the TGD4 protein was present in the outer envelope membrane of chloroplasts, where it appeared to be deeply buried within the membrane except for the N-terminus, which was found to be exposed to the cytosol. It is proposed that TGD4 is either directly involved in the transfer of polar lipids, possibly PtdOH, from the ER to the outer chloroplast envelope membrane or in the transfer of PtdOH through the outer envelope membrane.     

Chloroplast lipid transfer processes in Chlamydomonas reinhardtii involving a TRIGALACTOSYLDIACYLGLYCEROL 2 (TGD2) orthologue

In plants, lipids of the photosynthetic membrane are synthesized by parallel pathways associated with the endoplasmic reticulum (ER) and the chloroplast envelope membranes. Lipids derived from the two pathways are distinguished by their acyl‐constituents. Following this plant paradigm, the prevalent acyl composition of chloroplast lipids suggests that Chlamydomonas reinhardtii (Chlamydomonas) does not use the ER pathway; however, the Chlamydomonas genome encodes presumed plant orthologues of a chloroplast lipid transporter consisting of TGD (TRIGALACTOSYLDIACYLGLYCEROL) proteins that are required for ER‐to‐chloroplast lipid trafficking in plants. To resolve this conundrum, we identified a mutant of Chlamydomonas deleted in the TGD2 gene and characterized the respective protein, CrTGD2. Notably, the viability of the mutant was reduced, showing the importance of CrTGD2. Galactoglycerolipid metabolism was altered in the tgd2 mutant with monogalactosyldiacylglycerol (MGDG) synthase activity being strongly stimulated. We hypothesize this to be a result of phosphatidic acid accumulation in the chloroplast outer envelope membrane, the location of MGDG synthase in Chlamydomonas. Concomitantly, increased conversion of MGDG into triacylglycerol (TAG) was observed. This TAG accumulated in lipid droplets in the tgd2 mutant under normal growth conditions. Labeling kinetics indicate that Chlamydomonas can import lipid precursors from the ER, a process that is impaired in the tgd2 mutant.     

A small ATPase protein of Arabidopsis, TGD3, involved in chloroplast lipid import

Polar lipid trafficking is essential in eukaryotic cells as membranes of lipid assembly are often distinct from final destination membranes. A striking example is the biogenesis of the photosynthetic membranes (thylakoids) in plastids of plants. Lipid biosynthetic enzymes at the endoplasmic reticulum and the inner and outer plastid envelope membranes are involved. This compartmentalization requires extensive lipid trafficking. Mutants of Arabidopsis are available that are disrupted in the incorporation of endoplasmic reticulum-derived lipid precursors into thylakoid lipids. Two proteins affected in two of these mutants, trigalactosyldiacylglycerol 1 (TGD1) and TGD2, encode the permease and substrate binding component, respectively, of a proposed lipid translocator at the inner chloroplast envelope membrane. Here we describe a third protein of Arabidopsis, TGD3, a small ATPase proposed to be part of this translocator. As in the tgd1 and tgd2 mutants, triacylglycerols and trigalactolipids accumulate in a tgd3 mutant carrying a T-DNA insertion just 5' of the TGD3 coding region. The TGD3 protein shows basal ATPase activity and is localized inside the chloroplast beyond the inner chloroplast envelope membrane. Proteins orthologous to TGD1, -2, and -3 are predicted to be present in Gram- bacteria, and the respective genes are organized in operons suggesting a common biochemical role for the gene products. Based on the current analysis, it is hypothesized that TGD3 is the missing ATPase component of a lipid transporter involving TGD1 and TGD2 required for the biosynthesis of ER-derived thylakoid lipids in Arabidopsis.     

Homeostatic restitution of cell membranes. Nuclear membrane lipid biogenesis and transport of protein from cytosol to intranuclear spaces

Our studies on homeostatic restitution of cellular and subcellular membranes showed that vesicular intracellular transport is engaged in systematic and coordinated replacement of lipids and proteins in the membranes of the secretory, non-dividing epithelial cells (Slomiany et al., J. Physiol. Pharmacol. 2004; 55: 837-860). In this report, we present evidence on the homeostatic restitution of lipids in the biomembranes that constitute nuclear envelopes. We investigated nuclear membranes lipid synthesis by employing purified intact nuclei (IN), the outer nuclear membrane (ONM), the inner nuclear membrane (INM) and the cell cytosol (CC). In contrast to Endoplasmic Reticulum (ER) which in the presence of CC generates new biomembrane that forms ER vesicles transporting ER products to Golgi, the IN, ONM and INM are not producing transport vesicles. Instead, the newly synthesized lipids remain in the nuclear membranes. The membranes (INM, ONM) of IN incubated with CC become enriched with newly synthesized phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylinositol phosphates (PIPs) and phosphatidic acid (PA). The incubation of separated ONM and INM with CC also enriched the membranes with IN specific lipids identified above. Moreover, the incubation of IN or its membranes with CC afforded retention of numerous CC proteins on the nuclear membrane. Here, we concentrated on 30kDa CC protein that displayed affinity to nuclear membrane PIP2. The 30kDa CC protein bound to PIP2 of IN, INM, and ONM. With IN, initially the PIP2-30kDa CC protein complex was detected on ONM, after 30-120 min of incubation, was found on INM and in nuclear contents. At the same time when the 30 kDa protein was released from INM and found in nuclear contents, the PIP2 of INM and ONM became undetectable, while the lipid extract from the membrane displaced from IN contained labeled PI only. Since ONM is an uninterrupted continuum of ER and INM, we speculate that the synthesis of the lipids in the ER, in the region adjacent to nucleus, is defining nuclear outer and inner biomembrane composition, is responsible for transport of the cytosolic protein into the nucleus and, replenishment of ER membrane used for vesicular transport.     

The plastid outer membrane localized LPTD1 is important for glycerolipid remodelling under phosphate starvation

Glycerolipid synthesis in plants is coordinated between plastids and the endoplasmic reticulum (ER). A central step within the glycerolipid synthesis is the transport of phosphatidic acid from ER to chloroplasts. The chloroplast outer envelope protein TGD4 belongs to the LptD family conserved in bacteria and plants and selectively binds and may transport phosphatidic acid. We describe a second LptD‐family protein in A. thaliana (atLPTD1; At2g44640) characterized by a barrel domain with an amino‐acid signature typical for cyanobacterial LptDs. It forms a cation selective channel in vitro with a diameter of about 9 Å. atLPTD1 levels are induced under phosphate starvation. Plants expressing an RNAi construct against atLPTD1 show a growth phenotype under normal conditions. Expressing the RNAi against atLPTD1 in the tgd4–1 background renders the plants more sensitive to light stress or phosphate limitation than the individual mutants. Moreover, lipid analysis revealed that digalactosyldiacylglycerol and sulfoquinovosyldiacylglycerol levels remain constant in the RNAi mutants under phosphate starvation, while these two lipids are enhanced in wild‐type. Based on our results, we propose a function of atLPTD1 in the transport of lipids from ER to chloroplast under phosphate starvation, which is combinatory with the function of TGD4.     

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.     

A 25-Amino Acid Sequence of the Arabidopsis TGD2 Protein Is Sufficient for Specific Binding of Phosphatidic Acid

Genetic analysis suggests that the TGD2 protein of Arabidopsis is required for the biosynthesis of endoplasmic reticulum derived thylakoid lipids. TGD2 is proposed to be the substrate-binding protein of a presumed lipid transporter consisting of the TGD1 (permease) and TGD3 (ATPase) proteins. The TGD1, -2, and -3 proteins are localized in the inner chloroplast envelope membrane. TGD2 appears to be anchored with an N-terminal membrane-spanning domain into the inner envelope membrane, whereas the C-terminal domain faces the intermembrane space. It was previously shown that the C-terminal domain of TGD2 binds phosphatidic acid (PtdOH). To investigate the PtdOH binding site of TGD2 in detail, the C-terminal domain of the TGD2 sequence lacking the transit peptide and transmembrane sequences was fused to the C terminus of the Discosoma sp. red fluorescent protein (DR). This greatly improved the solubility of the resulting DR-TGD2C fusion protein following production in Escherichia coli. The DR-TGD2C protein bound PtdOH with high specificity, as demonstrated by membrane lipid-protein overlay and liposome association assays. Internal deletion and truncation mutagenesis identified a previously undescribed minimal 25-amino acid fragment in the C-terminal domain of TGD2 that is sufficient for PtdOH binding. Binding characteristics of this 25-mer were distinctly different from those of TGD2C, suggesting that additional sequences of TGD2 providing the proper context for this 25-mer are needed for wild type-like PtdOH binding.     

Mutation of the TGD1 chloroplast envelope protein affects phosphatidate metabolism in Arabidopsis

Phosphatidate (PA) is a central metabolite of lipid metabolism and a signaling molecule in many eukaryotes, including plants. Mutations in a permease-like protein, TRIGALACTOSYLDIACYLGLYCEROL1 (TGD1), in Arabidopsis thaliana caused the accumulation of triacylglycerols, oligogalactolipids, and PA. Chloroplast lipids were altered in their fatty acid composition consistent with an impairment of lipid trafficking from the endoplasmic reticulum (ER) to the chloroplast and a disruption of thylakoid lipid biosynthesis from ER-derived precursors. The process mediated by TGD1 appears to be essential as mutation of the protein caused a high incidence of embryo abortion. Isolated tgd1 mutant chloroplasts showed a decreased ability to incorporate PA into galactolipids. The TGD1 protein was localized to the inner chloroplast envelope and appears to be a component of a lipid transporter. As even partial disruption of TGD1 function has drastic consequences on central lipid metabolism, the tgd1 mutant provides a tool to explore regulatory mechanisms governing lipid homeostasis and lipid trafficking in plants.     

Three distinct lipid kinase activities are present in spinach chloroplast envelope membranes: phosphatidylinositol phosphorylation is sensitive to wortmannin and not dependent on chloroplast ATP.

Chloroplast envelope membranes display properties that are important in lipid synthesis, regulation of metabolites, and protein transport, as well as in signal transduction. The recent discovery showing that phosphorylation of lipids occurs in envelope membranes provides a new approach for understanding the role of chloroplast lipids in these processes. The present investigation shows that three major lipid kinase activities are at least present in envelope membranes. These activities greatly depend on external conditions, such as pH, ATP concentrations, temperature, and chloroplast ATP and wortmannin sensitivity. Two types of phosphorylated lipid couples displayed similar intrinsic responses toward these biochemical parameters, namely phosphatidic acid (PA) and its lysoderivative (LPA) and monogalactosyl-phosphate-diacylglycerol (MGpDG) and its lysoderivative (LMGpDG), but not phosphatidylinositol-monophosphate (PIP) and its lysoderivative (LPIP). Phosphorylation of phosphatidylinositol was not dependent on chloroplast ATP, but was sensitive toward wortmannin in intact chloroplasts and outer envelope membrane vesicles.     

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.     

Chloroplast lipid synthesis and lipid trafficking through ER-plastid membrane contact sites

Plant chloroplasts contain an intricate photosynthetic membrane system, the thylakoids, and are surrounded by two envelope membranes at which thylakoid lipids are assembled. The glycoglycerolipids mono- and digalactosyldiacylglycerol, and sulfoquinovosyldiacylglycerol as well as phosphatidylglycerol, are present in thylakoid membranes, giving them a unique composition. Fatty acids are synthesized in the chloroplast and are either directly assembled into thylakoid lipids at the envelope membranes or exported to the ER (endoplasmic reticulum) for extraplastidic lipid assembly. A fraction of lipid precursors is reimported into the chloroplast for the synthesis of thylakoid lipids. Thus polar lipid assembly in plants requires tight co-ordination between the chloroplast and the ER and necessitates inter-organelle lipid trafficking. In the present paper, we discuss the current knowledge of the export of fatty acids from the chloroplast and the import of chloroplast lipid precursors assembled at the ER. Direct membrane contact sites between the ER and the chloroplast outer envelopes are discussed as possible conduits for lipid transfer.     

Transport of phosphatidic acid within the mitochondrion

Transfer of phosphatidic acid from the outer to the inner membrane within intact rat liver mitochondria was assessed by measuring the ratio of lipid 32P to the marker enzyme of the outer membrane, rotenone-insensitive NADH-cytochrome c reductase, in the outer and inner membrane fractions obtained after incubation of mitochondria under conditions for net synthesis of [32P]phosphatidic acid. This transfer was found to proceed with time, to occur only under high ionic strength of the external medium and to be insensitive to N-ethylmaleimide and factors reducing the number of contact sites between the two mitochondrial membranes. These results are interpreted as supporting the idea that phosphatidic acid transport within the mitochondrion occurs as free diffusion through the aqueous phase and not being mediated by phospholipid transfer protein(s).     

Arabidopsis TRIGALACTOSYLDIACYLGLYCEROL5 Interacts with TGD1, TGD2, and TGD4 to Facilitate Lipid Transfer from the Endoplasmic Reticulum to Plastids

The biogenesis of photosynthetic membranes in the plastids of higher plants requires an extensive supply of lipid precursors from the endoplasmic reticulum (ER). Four TRIGALACTOSYLDIACYLGLYCEROL (TGD) proteins (TGD1,2,3,4) have thus far been implicated in this lipid transfer process. While TGD1, TGD2, and TGD3 constitute an ATP binding cassette transporter complex residing in the plastid inner envelope, TGD4 is a transmembrane lipid transfer protein present in the outer envelope. These observations raise questions regarding how lipids transit across the aqueous intermembrane space. Here, we describe the isolation and characterization of a novel Arabidopsis thaliana gene, TGD5. Disruption of TGD5 results in similar phenotypic effects as previously described in tgd1,2,3,4 mutants, including deficiency of ER-derived thylakoid lipids, accumulation of oligogalactolipids, and triacylglycerol. Genetic analysis indicates that TGD4 is epistatic to TGD5 in ER-to-plastid lipid trafficking, whereas double mutants of a null tgd5 allele with tgd1-1 or tgd2-1 show a synergistic embryo-lethal phenotype. TGD5 encodes a small glycine-rich protein that is localized in the envelope membranes of chloroplasts. Coimmunoprecipitation assays show that TGD5 physically interacts with TGD1, TGD2, TGD3, and TGD4. Collectively, these results suggest that TGD5 facilitates lipid transfer from the outer to the inner plastid envelope by bridging TGD4 with the TGD1,2,3 transporter complex.     

Enhanced thermal tolerance of photosynthesis and altered chloroplast ultrastructure in a mutant of Arabidopsis deficient in lipid desaturation

A mutant of Arabidopsis thaliana, deficient in activity of the chloroplast n-6 desaturase, accumulated high levels of C_16:1 and C_18:1 lipids and had correspondingly reduced levels of polyunsaturated lipids. The altered lipid composition of the mutant had pronounced effects on chloroplast ultrastructure, thylakoid membrane protein and chlorophyll content, electron transport rates, and the thermal stability of the photosynthetic membranes. The change in chloroplast ultrastructure was due to a 48% decrease in the amount of appressed membranes that was not compensated for by an increased amount of nonappressed membrane. This resulted in a net loss of 36% of the thylakoid membrane per chloroplast and a corresponding reduction in chlorophyll and protein content. Electrophoretic analysis of the chlorophyll-protein complexes further revealed a small decrease in the amount of light-harvesting complex. Relative levels of whole chain and protosystem II electron transport rates were also reduced in the mutant. In addition, the mutation resulted in enhanced thermal stability of photosynthetic electron transport. These observations suggest a central role of polyunsaturated lipids in determining chloroplast structure and maintaining normal photosynthetic function and demonstrate that lipid unsaturation directly affects the thermal stability of photosynthetic membranes.     

Glycerolipid transfer for the building of membranes in plant cells

Membranes of plant organelles have specific glycerolipid compositions. Selective distribution of lipids at the levels of subcellular organelles, membrane leaflets and membrane domains reflects a complex and finely tuned lipid homeostasis. Glycerolipid neosynthesis occurs mainly in plastid envelope and endoplasmic reticulum membranes. Since most lipids are not only present in the membranes where they are synthesized, one cannot explain membrane specific lipid distribution by metabolic processes confined in each membrane compartment. In this review, we present our current understanding of glycerolipid trafficking in plant cells. We examine the potential mechanisms involved in lipid transport inside bilayers and from one membrane to another. We survey lipid transfers going through vesicular membrane flow and those dependent on lipid transfer proteins at membrane contact sites. By introducing recently described membrane lipid reorganization during phosphate deprivation and recent developments issued from mutant analyses, we detail the specific lipid transfers towards or outwards the chloroplast envelope.     

Enhanced thermal tolerance in a mutant of Arabidopsis deficient in palmitic Acid unsaturation

A mutant of Arabidopsis thaliana, deficient in the activity of a chloroplast ω9 fatty acid desaturase, accumulates high amounts of palmitic acid (16:0), and exhibits an overall reduction in the level of unsaturation of chloroplast lipids. Under standard conditions the altered membrane lipid composition had only minor effects on growth rate of the mutant, net photosynthetic CO₂ fixation, photosynthetic electron transport, or chloroplast ultrastructure. Similarly, fluorescence polarization measurements indicated that the fluidity of the membranes was not significantly different in the mutant and the wild type. However, at temperatures above 28°C, the mutant grew more rapidly than the wild type suggesting that the altered fatty acid composition enhanced the thermal tolerance of the mutant. Similarly, the chloroplast membranes of the mutant were more resistant than wild type to thermal inactivation of photosynthetic electron transport. These observations lend support to previous suggestions that chloroplast membrane lipid composition may be an important component of the thermal acclimation response observed in many plant species which are photosynthetically active during periods of seasonally variable temperature extremes.     

The fusogenic lipid phosphatidic acid promotes the biogenesis of mitochondrial outer membrane protein Ugo1

Import and assembly of mitochondrial proteins depend on a complex interplay of proteinaceous translocation machineries. The role of lipids in this process has been studied only marginally and so far no direct role for a specific lipid in mitochondrial protein biogenesis has been shown. Here we analyzed a potential role of phosphatidic acid (PA) in biogenesis of mitochondrial proteins in Saccharomyces cerevisiae. In vivo remodeling of the mitochondrial lipid composition by lithocholic acid treatment or by ablation of the lipid transport protein Ups1, both leading to an increase of mitochondrial PA levels, specifically stimulated the biogenesis of the outer membrane protein Ugo1, a component of the mitochondrial fusion machinery. We reconstituted the import and assembly pathway of Ugo1 in protein-free liposomes, mimicking the outer membrane phospholipid composition, and found a direct dependency of Ugo1 biogenesis on PA. Thus, PA represents the first lipid that is directly involved in the biogenesis pathway of a mitochondrial membrane protein.     

Optical manipulation reveals strong attracting forces at membrane contact sites between endoplasmic reticulum and chloroplasts

Eukaryote cells depend on membrane lipid trafficking from biogenic membranes, like the endoplasmic reticulum (ER), to other membranes in the cell. Two major routes for membrane lipid transport are recognized: vesicular trafficking and lipid transfer at zones of close contact between membranes. Specific ER regions involved in such membrane contact sites (MCSs) have been isolated, and lipid transfer at MCSs as well as protein-protein interactions between the partaking membranes have been demonstrated (reviewed by Holthuis, J. C. M., and Levine, T. P. (2005) Nat. Rev. 6, 209-220). Here we present the first demonstration of the physical association between membranes involved in MCSs: by using optical imaging and manipulation, strong attracting forces between ER and chloroplasts are revealed. We used Arabidopsis thaliana expressing green fluorescent protein in the ER lumen and observed leaf protoplasts by confocal microscopy. The ER network was evident, with ER branch end points apparently localized at chloroplast surfaces. After rupture of a protoplast using a laser scalpel, the cell content was released. ER fragments remained attached to the released chloroplasts and could be stretched out by optical tweezers. The applied force, 400 pN, could not drag a chloroplast free from its attached ER, which could reflect protein-protein interactions at the ER-chloroplast MCSs. As chloroplasts rely on import of ER-synthesized lipids, we propose that lipid transfer occurs at these MCSs. We suggest that lipid transfer at the MCSs also occurs in the opposite direction, for example to channel plastid-synthesized acyl groups to supply substrates for ER-localized synthesis of membrane and storage lipids.