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.     

Lipids at membrane contact sites: cell signaling and ion transport

Communication between organelles is essential to coordinate cellular functions and the cell's response to physiological and pathological stimuli. Organellar communication occurs at membrane contact sites (MCSs), where the endoplasmic reticulum (ER) membrane is tethered to cellular organelle membranes by specific tether proteins and where lipid transfer proteins and cell signaling proteins are located. MCSs have many cellular functions and are the sites of lipid and ion transfer between organelles and generation of second messengers. This review discusses several aspects of MCSs in the context of lipid transfer, formation of lipid domains, generation of Ca²⁺ and cAMP second messengers, and regulation of ion transporters by lipids.     

Interorganellar communication and membrane contact sites in protozoan parasites

A key characteristic of eukaryotic cells is the presence of organelles with discrete boundaries and functions. Such subcellular compartmentalization into organelles necessitates platforms for communication and material exchange between each other which often involves vesicular trafficking and associated processes. Another way is via the close apposition between organellar membranes, called membrane contact sites (MCSs). Apart from lipid transfer, MCSs have been implicated to mediate in various cellular processes including ion transport, apoptosis, and organelle dynamics. In mammalian and yeast cells, contact sites have been reported between the membranes of the following: the endoplasmic reticulum (ER) and the plasma membrane (PM), ER and the Golgi apparatus, ER and endosomes (i.e., vacuoles, lysosomes), ER and lipid droplets (LD), the mitochondria and vacuoles, the nucleus and vacuoles, and the mitochondria and lipid droplets, whereas knowledge of MCSs in non-model organisms such as protozoan parasites is extremely limited. Growing evidence suggests that MCSs play more general and conserved roles in cell physiology. In this mini review, we summarize and discuss representative MCSs in divergent parasitic protozoa, and highlight the universality, diversity, and the contribution of MCSs to parasitism.     

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 role for lipid trafficking in chloroplast biogenesis

Chloroplasts are the defining plant organelle carrying out photosynthesis. Photosynthetic complexes are embedded into the thylakoid membrane which forms an intricate system of membrane lamellae and cisternae. The chloroplast boundary consists of two envelope membranes controlling the exchange of metabolites between the plastid and the extraplastidic compartments of the cell. The plastid internal matrix (stroma) is the primary location for fatty acid biosynthesis in plants. Fatty acids can be assembled into glycerolipids at the envelope membranes of plastids or they can be exported and assembled into lipids at the endoplasmic reticulum (ER) to provide building blocks for extraplastidic membranes. Some of these glycerolipids, assembled at the ER, return to the plastid where they are remodeled into the plastid typical glycerolipids. As a result of this cooperation of different subcellular membrane systems, a rich complement of lipid trafficking phenomena contributes to the biogenesis of chloroplasts. Considerable progress has been made in recent years towards a better mechanistic understanding of lipid transport across plastid envelopes. Lipid transporters of bacteria and plants have been discovered and their study begins to provide detailed mechanistic insights into lipid trafficking phenomena relevant to chloroplast biogenesis.     

PI4P/PS countertransport by ORP10 at ER–endosome membrane contact sites regulates endosome fission

Membrane contact sites (MCSs) serve as a zone for nonvesicular lipid transport by oxysterol-binding protein (OSBP)-related proteins (ORPs). ORPs mediate lipid countertransport, in which two distinct lipids are transported counterdirectionally. How such lipid countertransport controls specific biological functions, however, remains elusive. We report that lipid countertransport by ORP10 at ER–endosome MCSs regulates retrograde membrane trafficking. ORP10, together with ORP9 and VAP, formed ER–endosome MCSs in a phosphatidylinositol 4-phosphate (PI4P)-dependent manner. ORP10 exhibited a lipid exchange activity toward its ligands, PI4P and phosphatidylserine (PS), between liposomes in vitro, and between the ER and endosomes in situ. Cell biological analysis demonstrated that ORP10 supplies a pool of PS from the ER, in exchange for PI4P, to endosomes where the PS-binding protein EHD1 is recruited to facilitate endosome fission. Our study highlights a novel lipid exchange at ER–endosome MCSs as a nonenzymatic PI4P-to-PS conversion mechanism that organizes membrane remodeling during retrograde membrane trafficking.     

Pex30-like proteins function as adaptors at distinct ER membrane contact sites

Membrane lipids and proteins synthesized in the ER are used for de novo assembly of organelles, such as lipid droplets and peroxisomes. After assembly, the growth of these organelles is supported by ER-derived lipids transferred at membrane contact sites (MCSs). How ER sites for organelle biogenesis and lipid transfer are established and regulated is unclear. Here, we investigate how the ER membrane protein Pex30 and its family members Pex28, Pex29, Pex31, and Pex32 target and function at multiple MCSs. We show that different Pex30 complexes function at distinct ER domains and MCSs. Pex30 targets ER–peroxisome MCSs when bound to Pex28 and Pex32, organizes the nuclear–vacuolar junction when bound to Pex29, and promotes the biogenesis of lipid droplets independently of other family members. Importantly, the reticulon homology domain (RHD) mediates the assembly of the various Pex30 complexes. Given the role of RHD in membrane shaping, our findings offer a mechanistic link between MCS and regulation of membrane curvature.     

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.     

酵母线粒体的磷脂转运

线粒体是一种由两层膜包被的细胞器,其功能和结构的稳定性取决于线粒体膜上精确的磷脂组成及分布。线粒体膜上的大部分脂类物质由内质网合成,既而转运到线粒体。而部分脂类利用内质网上产生的前体,在线粒体内膜上合成。由此可见,线粒体膜脂的生物合成需要线粒体与内质网以及线粒体外膜(outer mitochondrial membrane, OMM)与内膜(inner mitochondrial membrane, IMM)之间进行大量的脂质转运。目前认为,这种运输过程既可在拴系因子(tether factors)形成的膜结合部位(membrane contact sites, MCSs)内发生,也可借助脂质转运蛋白(lipid transfer proteins, LTPs)完成。近年来,研究者以酵母为对象,建立了多种线粒体磷脂转运(phospholipid trafficking)的模型,这使人们初步理解了线粒体磷脂转运的机制。本综述总结了酵母线粒体磷脂转运的最新发现,并对这些磷脂转运的模型进行了讨论,以期为今后深入了解线粒体脂类代谢提供参考。     

Chlamydiae interaction with the endoplasmic reticulum: contact,function and consequences

Chlamydiae and chlamydiae‐related organisms are obligate intracellular bacterial pathogens. They reside in a membrane‐bound compartment termed the inclusion and have evolved sophisticated mechanisms to interact with cellular organelles. This review focuses on the nature, the function(s) and the consequences of chlamydiae–inclusion interaction with the endoplasmic reticulum (ER). The inclusion membrane establishes very close contact with the ER at specific sites termed ER–inclusion membrane contact sites (MCSs). These MCSs are constituted of a specific set of factors, including the C. trachomatis effector protein IncD and the host cell proteins CERT and VAPA/B. Because CERT and VAPA/B have a demonstrated role in the non‐vesicular trafficking of lipids between the ER and the Golgi, it was proposed that Chlamydia establish MCSs with the ER to acquire host lipids. However, the recruitment of additional factors to ER–inclusion MCSs, such as the ER calcium sensor STIM1, may suggest additional functions unrelated to lipid acquisition. Finally, chlamydiae interaction with the ER appears to induce the ER stress response, but this response is quickly dampened by chlamydiae to promote host cell survival.     

Short-range intracellular trafficking of small molecules across endoplasmic reticulum junctions

Intracellular trafficking is not mediated exclusively by vesicles. Additional, non-vesicular mechanisms transport material, in particular small molecules such as lipids and Ca(2+) ions, from one organelle to another. This transport occurs at narrow cytoplasmic gaps called membrane contact sites (MCSs), at which two organelles come into close apposition. Despite the conservation of these structures throughout evolution, little is known about this transport, largely because of a lack of knowledge of almost all molecular components of MCSs. Recently, this situation has started to change because the structural proteins that bridge an MCS are now known in a single case, and proteins implicated in lipid trafficking have been localized to MCSs. In the light of these advances, I hypothesize that the endoplasmic reticulum has a central role in the trafficking of lipids and ions by forming a network of MCSs with most other intracellular organelles.     

Lipid transfer and signaling at organelle contact sites: the tip of the iceberg

Membrane contact sites (MCSs) are formed by the close apposition of membranes of two organelles. They are zones where signals and small molecules, such as lipids and calcium, are exchanged between intracellular compartments. The past few years have seen considerable progress in our understanding of how MCSs form and facilitate the exchange of lipids and signals. Here we summarize what has been learned about MCSs between the endoplamic reticulum (ER) and the plasma membrane, the ER and mitochondria, and the ER and endosomes or lysosomes. These findings suggest that we are just beginning to understand how MCSs form and function.     

Organelle contacts: Sub‐organelle zones to facilitate rapid and accurate inter‐organelle trafficking of lipids

When one person wants to communicate securely with another, he/she should contact the other person directly. This rule applies not only to human society, but also to the intracellular micro‐society. In the past two decades, it has become increasingly clear that the sub‐organelle regions called membrane contact sites (MCSs) are pivotal for inter‐organelle transport of lipids in cells, as highlighted in the thematic review series “Interorganelle trafficking of lipids” held in Traffic in 2014–2015. In this commentary, we will describe how the currently prevailing model for lipid trafficking at MCSs was generated, and comment on three important issues that have not been explored: (a1) the principles guiding the generation of an asymmetrical inter‐organelle flow of lipids in cells, (b2) the advantages in lipid trafficking at organelle contacts, and (c3) the dynamic network of inter‐organelle lipid trafficking.     

Ultrastructural relationship of the phagophore with surrounding organelles

Phagophore nucleates from a subdomain of the endoplasmic reticulum (ER) termed the omegasome and also makes contact with other organelles such as mitochondria, Golgi complex, plasma membrane and recycling endosomes during its formation. We have used serial block face scanning electron microscopy (SB-EM) and electron tomography (ET) to image phagophore biogenesis in 3 dimensions and to determine the relationship between the phagophore and surrounding organelles at high resolution. ET was performed to confirm whether membrane contact sites (MCSs) are evident between the phagophore and those surrounding organelles. In addition to the known contacts with the ER, we identified MCSs between the phagophore and membranes from putative ER exit sites, late endosomes or lysosomes, the Golgi complex and mitochondria. We also show that one phagophore can have simultaneous MCSs with more than one organelle. Future membrane flux experiments are needed to determine whether membrane contacts also signify lipid translocation.     

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.     

Lipid synthesis and transport are coupled to regulate membrane lipid dynamics in the endoplasmic reticulum

Structural lipids are mostly synthesized in the endoplasmic reticulum (ER), from which they are actively transported to the membranes of other organelles. Lipids can leave the ER through vesicular trafficking or non-vesicular lipid transfer and, curiously, both processes can be regulated either by the transported lipid cargos themselves or by different secondary lipid species. For most structural lipids, transport out of the ER membrane is a key regulatory component controlling their synthesis. Distribution of the lipids between the two leaflets of the ER bilayer or between the ER and other membranes is also critical for maintaining the unique membrane properties of each cellular organelle. How cells integrate these processes within the ER depends on fine spatial segregation of the molecular components and intricate metabolic channeling, both of which we are only beginning to understand. This review will summarize some of these complex processes and attempt to identify the organizing principles that start to emerge. This article is part of a Special Issue entitled Endoplasmic reticulum platforms for lipid dynamics edited by Shamshad Cockcroft and Christopher Stefan.     

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.     

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

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

Piecing together the structural organisation of lipid exchange at membrane contact sites

Membrane contact sites (MCSs) are areas of close proximity between organelles, implicated in transport of small molecules and in organelle biogenesis. Lipid transfer proteins at MCSs facilitate the distribution of lipid species between organelle membranes. Such exchange processes rely on the apposition of two different membranes delimiting distinct compartments and a cytosolic intermembrane space. Maintaining organelle identity while transferring molecules therefore implies control over MCS architecture both on the ultrastructural and molecular levels. Factors including intermembrane distance, density of resident proteins, and contact surface area fine-tune MCS function. Furthermore, the structural arrangement of lipid transfer proteins and associated proteins underpins the molecular mechanisms of lipid fluxes at MCSs. Thus, the architecture of MCSs emerges as an essential aspect of their function.