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.     

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.     

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.     

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.     

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 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.     

Arabidopsis chloroplast lipid transport protein TGD2 disrupts membranes and is part of a large complex

In most plants the assembly of the photosynthetic thylakoid membrane requires lipid precursors synthesized at the endoplasmic reticulum (ER). Thus, the transport of lipids from the ER to the chloroplast is essential for biogenesis of the thylakoids. TGD2 is one of four proteins in Arabidopsis required for lipid import into the chloroplast, and was found to bind phosphatidic acid in vitro. However, the significance of phosphatidic acid binding for the function of TGD2 in vivo and TGD2 interaction with membranes remained unclear. Developing three functional assays probing how TGD2 affects lipid bilayers in vitro, we show that it perturbs membranes to the point of fusion, causes liposome leakage and redistributes lipids in the bilayer. By identifying and characterizing five new mutant alleles, we demonstrate that these functions are impaired in specific mutants with lipid phenotypes in vivo. At the structural level, we show that TGD2 is part of a protein complex larger than 500 kDa, the formation of which is disrupted in two mutant alleles, indicative of the biological relevance of this TGD2-containing complex. Based on the data presented, we propose that TGD2, as part of a larger complex, forms a lipid transport conduit between the inner and outer chloroplast envelope membranes, with its N terminus anchored in the inner membrane and its C terminus binding phosphatidic acid in the outer membrane.     

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.     

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.     

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.     

Protein import into cyanelles and complex chloroplasts

Higher-plant, green and red algal chloroplasts are surrounded by a double membrane envelope. The glaucocystophyte plastid (cyanelle) has retained a prokaryotic cell wall between the two envelope membranes. The complex chloroplasts of Euglena and dinoflagellates are surrounded by three membranes while the complex chloroplasts of chlorarachniophytes, cryptomonads, brown algae, diatoms and other chromophytes, are surrounded by 4 membranes. The peptidoglycan layer of the cyanelle envelope and the additional membranes of complex chloroplasts provide barriers to chloroplast protein import not present in the simpler double membrane chloroplast envelope. Analysis of presequence structure and in vitro import experiments indicate that proteins are imported directly from the cytoplasm across the two envelope membranes and peptidoglycan layer into cyanelles. Protein import into complex chloroplasts is however fundamentally different. Analysis of presequence structure and in vitro import into microsomal membranes has shown that translocation into the ER is the first step for protein import into complex chloroplasts enclosed by three or four membranes. In vivo pulse chase experiments and immunoelectronmicroscopy have shown that in Euglena, proteins are transported from the ER to the Golgi apparatus prior to import across the three chloroplast membranes. Ultrastructural studies and the presence of ribosomes on the outermost of the four envelope membranes suggests protein import into 4 membrane-bounded complex chloroplasts is directly from the ER like outermost membrane into the chloroplast. The fundamental difference in import mechanisms, post-translational direct chloroplast import or co-translational translocation into the ER prior to chloroplast import, appears to reflect the evolutionary origin of the different chloroplast types. Chloroplasts with a two-membrane envelope are thought to have evolved through the primary endosymbiotic association between a eukaryotic host and a photosynthetic prokaryote while complex chloroplasts are believed to have evolved through a secondary endosymbiotic association between a heterotrophic or possibly phototrophic eukaryotic host and a photosynthetic eukaryote.     

A NOVEL BIOFOULING ASSAY USING COMPUTER-ASSISTED MOTION ANALYSIS OF HINCKSIA IRREGULARIS SPORE SWIMMING

Chloroplasts in heterokont algae probably originated from a red algal endosymbiont which was engulfed and retained by a eukaryotic host, and are surrounded by four envelope membranes. The outermost of these membranes is called chloroplast ER (CER) and usually connects with the nuclear envelope. This information, however, is based mainly on studies on single-plastid heterokont algae. In multi-plastid heterokont algae, it is still unclear whether CER is continuous with the nuclear envelope. Since nuclear-encoded chloroplast proteins are synthesized by ribosomes on the ER membrane, clarifying the ER-CER structure in the heterokont algae is important in order to know the targeting pathway of those proteins. We did a detailed ultrastructural observation of endomembrane systems in a multi-plastid heterokont alga: Heterosigma akashiwo , and confirmed that the CER membrane was continuous with the ER membrane. However, unlike the CER membranes in other heterokont algae, it seemed to have very few ribosome attached. We also performed experiments for protein targeting into canine microsomes using a precursor for a nuclear-encoded chloroplast protein, a fucoxanthin-chlorophyll protein (FCP), of H. akashiwo , to see if the protein is targeted to the ER. It demonstrated that the precursor has a functional signal sequence for ER targeting, and is co-translationally translocated into the microsomes. Based on these data, we propose a hypothesis that, in H. akashiwo , nuclear-encoded chloroplast protein precursors that have been co-translationally inserted into the ER lumen are sorted in the ER and transported to the chloroplasts through the ER.     

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.     

Identification and characterization of Cor413im proteins as novel components of the chloroplast inner envelope

Plastids are surrounded by two membrane layers, the outer and inner envelope membranes, which have various transport and metabolic activities. A number of envelope membrane proteins have been identified by biochemical approaches and have been assigned to specific functions. Despite those efforts, the chloroplast envelope membrane is expected to contain a number of as yet unidentified proteins that may affect specific aspects of plant growth and development. In this report, we identify and characterize a novel class of inner envelope membrane proteins, designated as Cor413 chloroplast inner envelope membrane group (Cor413im). Both in vivo and in vitro studies indicate that Cor413im proteins are targeted to the chloroplast envelope. Biochemical analyses of Cor413im1 demonstrate that it is an integral membrane protein in the inner envelope of chloroplasts. Quantitative real-time PCR analysis reveals that COR413IM1 is more abundant than COR413IM2 in cold-acclimated Arabidopsis leaves. The analyses of T-DNA insertion mutants indicate that a single copy of COR413IM genes is sufficient to provide normal freezing tolerance to Arabidopsis. Based on these data, we propose that Cor413im proteins are novel components that are targeted to the chloroplast inner envelope in response to low temperature.     

ENDOMEMBRANE STRUCTURE AND THE CHLOROPLAST PROTEIN TARGETING PATHWAY IN HETEROSIGMA AKASHIWO (RAPHIDOPHYCEAE, CHROMISTA)

Chloroplasts in heterokont algae are surrounded by four membranes and probably originated from a red algal endosymbiont that was engulfed and retained by eukaryotic host. Understanding how nuclear-encoded chloroplast proteins are translocated from the cytoplasm into the chloroplast across these membranes could give us some insights about how the endosymbiont was integrated into the host cell in the process of secondary symbiogenesis. In multiplastid heterokont algae such as raphidophytes, it has been unclear if the outermost of the four membranes surrounding the chloroplast (the chloroplast endoplasmic reticulum [CER] membrane) is continuous with the nuclear envelope and rough endoplasmic reticulum (ER). Here, we report detailed ultrastructural observations of the raphidophyte Heterosigma akashiwo (Hada) Hada ex Y. Hara et Chihara that show that the CER membranes were continuous with ER membranes that had attached ribosomes, implying that the chloroplast with three envelope membranes is located within the ER lumen, that is, topologically the same structure as that of monoplastid heterokont algae. However, the CER membrane of H. akashiwo had very few, if any, ribosomes attached, unlike the CER membranes in other heterokont algae. To verify that proteins are first targeted to the ER, we assayed protein import into canine microsomes using a precursor for a nuclear-encoded chloroplast protein, the fucoxanthin-chlorophyll a / c protein of H. akashiwo. This demonstrated that the precursor has a functional signal sequence for ER targeting and is cotranslationally translocated into the ER, where a signal sequence of about 17 amino acids is removed. Based on these data, we hypothesize that in H. akashiwo , nuclear-encoded chloroplast protein precursors that have been cotranslationally transported into the ER lumen are sorted in the ER and transported to the chloroplasts through the ER lumen.     

Analysis of pea chloroplast inner and outer envelope membrane proteins by two-dimensional gel electrophoresis and their comparison with stromal proteins

Analysis of inner and outer pea (Pisum sativum var. Laxtons Progress No. 9) chloroplast envelope membranes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that, although the two membranes have distinct polypeptide compositions, there are several comigrating polypeptides in the two membrane fractions. To determine whether these comigrating polypeptides were identical by criteria other than molecular weight, the membrane proteins were analyzed by two-dimensional gel electrophoresis. The results demonstrated that an 86-kilodalton band found in both membranes represents at least two different polypeptides, one an outer membrane protein and the other an inner membrane protein. Several other polypeptide bands found in both membranes appear to be of stromal origin. Two of these polypeptides were shown to be the large and small subunits of ribulose 1,5-bisphosphate carboxylase. The large subunit was identified by two-dimensional electrophoresis of envelope membranes to which stromal proteins were added. Additionally, the large and small subunits of ribulose 1,5-bisphosphate carboxylase were immunologically identified using an electrophoretic transfer procedure coupled with an enzyme-linked immunosorbent assay. Various treatments, including sonication, resulted in no significant loss of the stromal polypeptides from the outer envelope membranes. Based on these results, it is suggested that the stromal proteins are not simply bound to the outer surface of the vesicles.     

ENDOMEMBRANE ULTRASTRUCTURE AND POSSIBLE CHLOROPLAST PROTEIN IMPORT PATHWAY IN HETEROSIGMA AKASHIWO (RAPHIDOPHYCEAE)

Chloroplasts in heterokont algae probably originated from a red algal endosymbiont which was engulfed and retained by a eukaryotic host, and are surrounded by four envelope membranes. The outermost of these membranes is called chloroplast ER (CER) and usually connects with the nuclear envelope. This information, however, is based mainly on studies on single‐plastid heterokont algae. In multi‐plastid heterokont algae, it is still unclear whether CER is continuous with the nuclear envelope. Since nuclear‐encoded chloroplast proteins are synthesized by ribosomes on the ER membrane, clarifying the ER‐CER structure in the heterokont algae is important in order to know the targeting pathway of those proteins. We did a detailed ultrastructural observation of endomembrane systems in a multi‐plastid heterokont alga: Heterosigma akashiwo, and confirmed that the CER membrane was continuous with the ER membrane. However, unlike the CER membranes in other heterokont algae, it seemed to have very few ribosome attached. We also performed experiments for protein targeting into canine microsomes using a precursor for a nuclear‐encoded chloroplast protein, a fucoxanthin‐chlorophyll protein (FCP), of H. akashiwo, to see if the protein is targeted to the ER. It demonstrated that the precursor has a functional signal sequence for ER targeting, and is co‐translationally translocated into the microsomes. Based on these data, we propose a hypothesis that, in H. akashiwo, nuclear‐encoded chloroplast protein precursors that have been co‐translationally inserted into the ER lumen are sorted in the ER and transported to the chloroplasts through the ER.     

A new chloroplast protein import intermediate reveals distinct translocation machineries in the two envelope membranes: energetics and mechanistic implications

Chloroplast protein import presents a complex membrane traversal problem: precursor proteins must cross two envelope membranes to reach the stromal compartment. This work characterizes a new chloroplast protein import intermediate which has completely traversed the outer envelope membrane but has not yet reached the stroma. The existence of this intermediate demonstrates that distinct protein transport machineries are present in both envelope membranes, and that they are able to operate independently of one another under certain conditions. Energetic characterization of this pathway led to the identification of three independent energy-requiring steps: binding of the precursor to the outer envelope membrane, outer membrane transport, and inner membrane transport. Localization of the sites of energy utilization for each of these steps, as well as their respective nucleotide specificities, suggest that three different ATPases mediate chloroplast envelope transport.     

Specific lipids influence the import capacity of the chloroplast outer envelope precursor protein translocon

Recent studies demonstrated that lipids influence the assembly and efficiency of membrane-embedded macromolecular complexes. Similarly, lipids have been found to influence chloroplast precursor protein binding to the membrane surface and to be associated with the Translocon of the Outer membrane of Chloroplasts (TOC). We used a system based on chloroplast outer envelope vesicles from Pisum sativum to obtain an initial understanding of the influence of lipids on precursor protein translocation across the outer envelope. The ability of the model precursor proteins p(OE33)titin and pSSU to be recognized and translocated in this simplified system was investigated. We demonstrate that transport across the outer membrane can be observed in the absence of the inner envelope translocon. The translocation, however, was significantly slower than that observed for chloroplasts. Enrichment of outer envelope vesicles with different lipids natively found in chloroplast membranes altered the binding and transport behavior. Further, the results obtained using outer envelope vesicles were consistent with the results observed for the reconstituted isolated TOC complex. Based on both approaches we concluded that the lipids sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylinositol (PI) increased TOC-mediated binding and import for both precursor proteins. In contrast, enrichment in digalactosyldiacylglycerol (DGDG) improved TOC-mediated binding for pSSU, but decreased import for both precursor proteins. Optimal import occurred only in a narrow concentration range of DGDG.     

Prediction of the plant beta-barrel proteome: a case study of the chloroplast outer envelope

In the postgenomic era, the transformation of genetic information into biochemical meaning is required. We have analyzed the proteome of the chloroplast outer envelope membrane by an in silico and a proteomic approach. Based on its evolutionary relation to the outer membrane of Gram-negative bacteria, the outer envelope membrane should contain a large number of beta-barrel proteins. We therefore calculated the probability for the existence of beta-sheet, beta-barrel, and hairpin structures among all proteins of the Arabidopsis thaliana genome. According to the existence of these structures, a number of candidates were selected. This protein pool was analyzed by TargetP to discard sequences with signals that would direct the protein to other organelles different from chloroplasts. In addition, the pool was manually controlled for the presence of proteins known to function outside of the chloroplast envelope. The approach developed here can be used to predict the topology of beta-barrel proteins. For the proteomic approach, proteins of highly purified outer envelope membranes of chloroplasts from Pisum sativum were analyzed by ESI-MS/MS mass spectrometry. In addition to the known components, four new proteins of the outer envelope membranes were identified in this study.