LDIP cooperates with SEIPIN and LDAP to facilitate lipid droplet biogenesis in Arabidopsis

Cytoplasmic lipid droplets (LDs) are evolutionarily conserved organelles that store neutral lipids and play critical roles in plant growth, development, and stress responses. However, the molecular mechanisms underlying their biogenesis at the endoplasmic reticulum (ER) remain obscure. Here we show that a recently identified protein termed LD-associated protein [LDAP]-interacting protein (LDIP) works together with both endoplasmic reticulum-localized SEIPIN and the LD-coat protein LDAP to facilitate LD formation in Arabidopsis thaliana. Heterologous expression in insect cells demonstrated that LDAP is required for the targeting of LDIP to the LD surface, and both proteins are required for the production of normal numbers and sizes of LDs in plant cells. LDIP also interacts with SEIPIN via a conserved hydrophobic helix in SEIPIN and LDIP functions together with SEIPIN to modulate LD numbers and sizes in plants. Further, the co-expression of both proteins is required to restore normal LD production in SEIPIN-deficient yeast cells. These data, combined with the analogous function of LDIP to a mammalian protein called LD Assembly Factor 1, are discussed in the context of a new model for LD biogenesis in plant cells with evolutionary connections to LD biogenesis in other eukaryotes.

The lipid droplet (LD) proteins LDIP and LDAP cooperate with endoplasmic reticulum-localized SEIPIN to coordinate LD formation in plant cells.     

Dynamics of protein and polar lipid recruitment during lipid droplet assembly in Chlamydomonas reinhardtii

In plants, neutral lipids are frequently synthesized and stored in seed tissues, where the assembly of lipid droplets (LDs) coincides with the accumulation of triacylglycerols (TAGs). In addition, photosynthetic, vegetative cells can form cytosolic LDs and much less information is known about the makeup and biogenesis of these LDs. Here we focus on Chlamydomonas reinhardtii as a reference model for LDs in a photosynthetic cell, because in this unicellular green alga LD dynamics can be readily manipulated by nitrogen availability. Nitrogen deprivation leads to cellular quiescence during which cell divisions cease and TAGs accumulate. The major lipid droplet protein (MLDP) forms a proteinaceous coat surrounding mature LDs. Reducing the amount of MLDP affects LD size and number, TAG breakdown and timely progression out of cellular quiescence following nitrogen resupply. Depending on nitrogen availability, MLDP recruits different proteins to LDs, tubulins in particular. Conversely, depolymerization of microtubules drastically alters the association of MLDP with LDs. LDs also contain select chloroplast envelope membrane proteins hinting at an origin of LDs, at least in part, from chloroplast membranes. Moreover, LD surface lipids are rich in de novo synthesized fatty acids, and are mainly composed of galactolipids which are typical components of chloroplast membranes. The composition of the LD membrane is altered in the absence of MLDP. Collectively, our results suggest a mechanism for LD formation in C. reinhardtii involving chloroplast envelope membranes by which specific proteins are recruited to LDs and a specialized polar lipid monolayer surrounding the LD is formed.     

Mouse fat storage‐inducing transmembrane protein 2 (FIT2) promotes lipid droplet accumulation in plants

Fat storage‐inducing transmembrane protein 2 (FIT2) is an endoplasmic reticulum (ER)‐localized protein that plays an important role in lipid droplet (LD) formation in animal cells. However, no obvious homologue of FIT2 is found in plants. Here, we tested the function of FIT2 in plant cells by ectopically expressing mouse (Mus musculus) FIT2 in Nicotiana tabacum suspension‐cultured cells, Nicotiana benthamiana leaves and Arabidopsis thaliana plants. Confocal microscopy indicated that the expression of FIT2 dramatically increased the number and size of LDs in leaves of N. benthamiana and Arabidopsis, and lipidomics analysis and mass spectrometry imaging confirmed the accumulation of neutral lipids in leaves. FIT2 also increased seed oil content by ~13% in some stable, overexpressing lines of Arabidopsis. When expressed transiently in leaves of N. benthamiana or suspension cells of N. tabacum, FIT2 localized specifically to the ER and was often concentrated at certain regions of the ER that resembled ER‐LD junction sites. FIT2 also colocalized at the ER with other proteins known to be involved in triacylglycerol biosynthesis or LD formation in plants, but not with ER resident proteins involved in electron transfer or ER‐vesicle exit sites. Collectively, these results demonstrate that mouse FIT2 promotes LD accumulation in plants, a surprising functional conservation in the context of a plant cell given the apparent lack of FIT2 homologues in higher plants. These results suggest also that FIT2 expression represents an effective synthetic biology strategy for elaborating neutral lipid compartments in plant tissues for potential biofuel or bioproduct purposes.     

Visualization of lipid droplet composition by direct organelle mass spectrometry

An expanding appreciation for the varied functions of neutral lipids in cellular organisms relies on a more detailed understanding of the mechanisms of lipid production and packaging into cytosolic lipid droplets (LDs). Conventional lipid profiling procedures involve the analysis of tissue extracts and consequently lack cellular or subcellular resolution. Here, we report an approach that combines the visualization of individual LDs, microphase extraction of lipid components from droplets, and the direct identification of lipid composition by nanospray mass spectrometry, even to the level of a single LD. The triacylglycerol (TAG) composition of LDs from several plant sources (mature cotton (Gossypium hirsutum) embryos, roots of cotton seedlings, and Arabidopsis thaliana seeds and leaves) were examined by direct organelle mass spectrometry and revealed the heterogeneity of LDs derived from different plant tissue sources. The analysis of individual LDs makes possible organellar resolution of molecular compositions and will facilitate new studies of LD biogenesis and functions, especially in combination with analysis of morphological and metabolic mutants. Furthermore, direct organelle mass spectrometry could be applied to the molecular analysis of other subcellular compartments and macromolecules.     

Proteomic study and marker protein identification of Caenorhabditis elegans lipid droplets

Lipid droplets (LDs) are a neutral lipid storage organelle that is conserved across almost all species. Many metabolic syndromes are directly linked to the over-storage of neutral lipids in LDs. The study of LDs in Caenorhabditis elegans (C. elegans) has been difficult because of the lack of specific LD marker proteins. Here we report the purification and proteomic analysis of C. elegans lipid droplets for the first time. We identified 306 proteins, 63% of these proteins were previously known to be LD-proteins, suggesting a similarity between mammalian and C. elegans LDs. Using morphological and biochemical analyses, we show that short-chain dehydrogenase, DHS-3 is almost exclusively localized on C. elegans LDs, indicating that it can be used as a LD marker protein in C. elegans. These results will facilitate further mechanistic studies of LDs in this powerful genetic system, C. elegans.     

A lipid droplet protein of Nannochloropsis with functions partially analogous to plant oleosins

As our understanding of the dynamics of lipid droplets (LDs) in animal, plant, and fungal cells is rapidly evolving, still little is known about the formation and turnover of these organelles in microalgae. Yet with the growing importance of algal feedstock for the production of biofuels and high-value lipids, there is a need to understand the mechanisms of LD dynamics in microalgae. Thus, we investigated the proteins associated with LDs of the emerging heterokont model alga Nannochloropsis sp. and discovered an abundant hydrophobic lipid droplet surface protein (LDSP) with unique primary sequence but structural similarities to other LD proteins. LDSP abundance in Nannochloropsis cells closely tracked the amount of triacylglycerols during conditions of oil accumulation and degradation. Functional characterization of LDSP in an Arabidopsis (Arabidopsis thaliana) OLEOSIN1-deficient mutant allowed a separation of its physical and structural properties in its interaction with LDs from its physiological or biochemical activities. Although LDSP presence in Arabidopsis predictably affected LD size, it could not reverse the physiological impact of OLEOSIN deficiency on triacylglycerol hydrolysis during germination.     

CIDE family proteins control lipid homeostasis and the development of metabolic diseases

Dysregulation of lipid homeostasis leads to the development of metabolic disorders including obesity, diabetes, cardiovascular disease and cancer. Lipid droplets (LDs) are subcellular organelles vital in the maintenance of lipid homeostasis by coordinating lipid synthesis, lipid storage, lipid secretion and lipolysis. Under fed condition, free fatty acids (FFAs) are remodeled and esterified into neutral lipids by lipogenesis and stored in the LDs. The lipid storage capacity of LDs is controlled by its growth via local lipid synthesis or by LD fusion. During fasting, neutral lipids are hydrolyzed by lipolysis, released as FFAs and secreted to meet energy demand. C ell death‐i nducing D NA fragmentation factor alpha (DFFA)‐like e ffector (CIDE) family proteins composed of Cidea, Cideb and Cidec/Fsp27 are ER‐ and LD‐associated proteins and have emerged as important regulators of lipid homeostasis. Notably, when localized on the LDs, CIDE proteins enrich at the LD‐LD contact sites (LDCSs) and control LD fusion and growth. Here, we summarize these recent advances made on the role of CIDE proteins in the regulation of lipid metabolism with a particular focus on the molecular mechanisms underlying CIDE‐mediated LD fusion and growth.     

Seipin regulates ER–lipid droplet contacts and cargo delivery

Seipin is an endoplasmic reticulum (ER) membrane protein implicated in lipid droplet (LD) biogenesis and mutated in severe congenital lipodystrophy (BSCL2). Here, we show that seipin is stably associated with nascent ER–LD contacts in human cells, typically via one mobile focal point per LD. Seipin appears critical for such contacts since ER–LD contacts were completely missing or morphologically aberrant in seipin knockout and BSCL2 patient cells. In parallel, LD mobility was increased and protein delivery from the ER to LDs to promote LD growth was decreased. Moreover, while growing LDs normally acquire lipid and protein constituents from the ER, this process was compromised in seipin‐deficient cells. In the absence of seipin, the initial synthesis of neutral lipids from exogenous fatty acid was normal, but fatty acid incorporation into neutral lipids in cells with pre‐existing LDs was impaired. Together, our data suggest that seipin helps to connect newly formed LDs to the ER and that by stabilizing ER–LD contacts seipin facilitates the incorporation of protein and lipid cargo into growing LDs in human cells.     

Seipin collaborates with the ER membrane to control the sites of lipid droplet formation

Most cells store metabolic energy in lipid droplets (LDs). LDs are composed of a hydrophobic core, covered by a phospholipid monolayer, and functionalized by a specific set of proteins. Formation of LDs takes place in the endoplasmic reticulum (ER), where neutral lipid biosynthetic enzymes are located. Recent evidence indicate that this process is confined to specific ER subdomains, where proteins meet to initiate LD assembly. The lipodystrophy protein Seipin, is emerging as a major coordinator of LD biogenesis. Seipin forms a large oligomeric toroidal structure, which traps neutral lipids to promote LD nucleation. Here, we discuss the role of LD biogenesis factors that associate with Seipin to assemble functional LDs.     

Small rubber particle proteins from Taraxacum brevicorniculatum promote stress tolerance and influence the size and distribution of lipid droplets and artificial poly(cis‐1,4‐isoprene) bodies

Natural rubber biosynthesis occurs on rubber particles, i.e. organelles resembling small lipid droplets localized in the laticifers of latex‐containing plant species, such as Hevea brasiliensis and Taraxacum brevicorniculatum. The latter expresses five small rubber particle protein (SRPP) isoforms named TbSRPP1–5, the most abundant proteins in rubber particles. These proteins maintain particle stability and are therefore necessary for rubber biosynthesis. TbSRPP1–5 were transiently expressed in Nicotiana benthamiana protoplasts and the proteins were found to be localized on lipid droplets and in the endoplasmic reticulum, with TbSRPP1 and TbSRPP3 also present in the cytosol. Bimolecular fluorescence complementation confirmed pairwise interactions between all proteins except TbSRPP2. The corresponding genes showed diverse expression profiles in young T. brevicorniculatum plants exposed to abiotic stress, and all except TbSRPP4 and TbSRPP5 were upregulated. Young Arabidopsis thaliana plants that overexpressed TbSRPP2 and TbSRPP3 tolerated drought stress better than wild‐type plants. Furthermore, we used rubber particle extracts and standards to investigate the affinity of the TbSRPPs for different phospholipids, revealing a preference for negatively charged head groups and 18:2/16:0 fatty acid chains. This finding may explain the effect of TbSRPP3–5 on the dispersity of artificial poly(cis‐1,4‐isoprene) bodies and on the lipid droplet distribution we observed in N. benthamiana leaves. Our data provide insight into the assembly of TbSRPPs on rubber particles, their role in rubber particle structure, and the link between rubber biosynthesis and lipid droplet‐associated stress responses, suggesting that SRPPs form the basis of evolutionarily conserved intracellular complexes in plants.     

Identification and characterization of associated with lipid droplet protein 1: A novel membrane-associated protein that resides on hepatic lipid droplets

Alcoholic and nonalcoholic liver steatosis and steatohepatitis are characterized by the massive accumulation of lipid droplets (LDs) in the cytosol of hepatocytes. Although LDs are ubiquitous and dynamic organelles found in the cells of a wide range of organisms, little is known about the mechanisms and sites of LD biogenesis. To examine the participation of these organelles in the pathophysiological disorders of steatotic livers, we used a combination of mass spectrometry (matrix-assisted laser desorption ionization-time of flight and LC-MS electrospray) and Western blot analysis to study the composition of LDs purified from rat liver after a partial hepatectomy. Fifty proteins were identified. Adipose differentiation-related protein was the most abundant, but other proteins such as calreticulin, TIP47, Sar1, Rab GTPases, Rho and actin were also found. In addition, we identified protein associated with lipid droplets I ALDI (tentatively named Associated with LD protein 1), a novel protein widely expressed in liver and kidney corresponding to the product of 0610006F02Rik (GI:27229118). Our results show that, upon lipid loading of the cells, ALDI translocates from the endoplasmic reticulum into nascent LDs and indicate that ALDI may be targeted to the initial lipid deposits that eventually form these droplets. Moreover, we used ALDI expression studies to view other processes related to these droplets, such as LD biogenesis, and to analyze LD dynamics. In conclusion, here we report the composition of hepatic LDs and describe a novel bona fide LD-associated protein that may provide new insights into the mechanisms and sites of LD biogenesis.     

Ancient ubiquitous protein 1 (AUP1) localizes to lipid droplets and binds the E2 ubiquitin conjugase G2 (Ube2g2) via its G2 binding region

Lipid droplets (LDs), the major intracellular storage sites for neutral lipids, consist of a neutral lipid core surrounded by a phospholipid monolayer membrane. In addition to their function in lipid storage, LDs participate in lipid biosynthesis and recently were implicated in proteasomal protein degradation and autophagy. To identify components of the protein degradation machinery on LDs, we studied several candidates identified in previous LD proteome analyses. Here, we demonstrate that the highly conserved and broadly expressed ancient ubiquitous protein 1 (AUP1) localizes to LDs, where it integrates into the LD surface in a monotopic fashion with both termini facing the cytosol. AUP1 contains a C-terminal domain with strong homology to a domain known as G2BR, which binds E2 ubiquitin conjugases. We show that AUP1, by means of its G2BR domain, binds to Ube2g2. This binding is abolished by deletion or mutation of the G2BR domain, although the LD localization of AUP1 is not affected. The presence of the AUP1-Ube2g2 complex at LDs provides a direct molecular link between LDs and the cellular ubiquitination machinery.     

Biogenesis and functions of lipid droplets in plants: Thematic Review Series: Lipid Droplet Synthesis and Metabolism: from Yeast to Man

The compartmentation of neutral lipids in plants is mostly associated with seed tissues, where triacylglycerols (TAGs) stored within lipid droplets (LDs) serve as an essential physiological energy and carbon reserve during postgerminative growth. However, some nonseed tissues, such as leaves, flowers and fruits, also synthesize and store TAGs, yet relatively little is known about the formation or function of LDs in these tissues. Characterization of LD-associated proteins, such as oleosins, caleosins, and sterol dehydrogenases (steroleosins), has revealed surprising features of LD function in plants, including stress responses, hormone signaling pathways, and various aspects of plant growth and development. Although oleosin and caleosin proteins are specific to plants, LD-associated sterol dehydrogenases also are present in mammals, and in both plants and mammals these enzymes have been shown to be important in (steroid) hormone metabolism and signaling. In addition, several other proteins known to be important in LD biogenesis in yeasts and mammals are conserved in plants, suggesting that at least some aspects of LD biogenesis and/or function are evolutionarily conserved.     

Characterization of stress‐responsive lncRNAs in Arabidopsis thaliana by integrating expression,epigenetic and structural features

Agrobacterium genetically transforms plants by transferring and integrating T‐(transferred) DNA into the host genome. This process requires both Agrobacterium and host proteins. VirE2 interacting protein 1 (VIP1), an Arabidopsis bZIP protein, has been suggested to mediate transformation through interaction with and targeting of VirE2 to nuclei. We examined the susceptibility of Arabidopsis vip1 mutant and VIP1 overexpressing plants to transformation by numerous Agrobacterium strains. In no instance could we detect altered transformation susceptibility. We also used confocal microscopy to examine the subcellular localization of Venus‐tagged VirE2 or Venus‐tagged VIP1, in the presence or absence of the other untagged protein, in different plant cell systems. We found that VIP1–Venus localized in both the cytoplasm and the nucleus of Arabidopsis roots, agroinfiltrated Nicotiana benthamiana leaves, Arabidopsis mesophyll protoplasts and tobacco BY‐2 protoplasts, regardless of whether VirE2 was co‐expressed. VirE2 localized exclusively to the cytoplasm of tobacco and Arabidopsis protoplasts, whether in the absence or presence of VIP1 overexpression. In transgenic Arabidopsis plants and agroinfiltrated N. benthamina leaves we could occasionally detect small aggregates of the Venus signal in nuclei, but these were likely to be imagining artifacts. The vast majority of VirE2 remained in the cytoplasm. We conclude that VIP1 is not important for Agrobacterium‐mediated transformation or VirE2 subcellular localization.     

The nuclear protein Poly(ADP‐ribose) polymerase 3 (AtPARP3) is required for seed storability in Arabidopsis thaliana

The deterioration of seeds during prolonged storage results in a reduction of viability and germination rate. DNA damage is one of the major cellular defects associated with seed deterioration. It is provoked by the formation of reactive oxygen species (ROS) even in the quiescent state of the desiccated seed. In contrast to other stages of seed life, DNA repair during storage is hindered through the low seed water content; thereby DNA lesions can accumulate. To allow subsequent seedling development, DNA repair has thus to be initiated immediately upon imbibition. Poly(ADP‐ribose) polymerases (PARPs) are important components in the DNA damage response in humans. Arabidopsis thaliana contains three homologues to the human HsPARP1 protein. Of these three, only AtPARP3 was very highly expressed in seeds. Histochemical GUS staining of embryos and endosperm layers revealed strong promoter activity of AtPARP3 during all steps of germination. This coincided with high ROS activity and indicated a role of the nuclear‐localised AtPARP3 in DNA repair during germination. Accordingly, stored parp3‐1 mutant seeds lacking AtPARP3 expression displayed a delay in germination as compared to Col‐0 wild‐type seeds. A controlled deterioration test showed that the mutant seeds were hypersensitive to unfavourable storage conditions. The results demonstrate that AtPARP3 is an important component of seed storability and viability.     

ATGL and CGI-58 are lipid droplet proteins of the hepatic stellate cell line HSC-T6

Lipid droplets (LDs) of hepatic stellate cells (HSCs) contain large amounts of vitamin A [in the form of retinyl esters (REs)] as well as other neutral lipids such as TGs. During times of insufficient vitamin A availability, RE stores are mobilized to ensure a constant supply to the body. To date, little is known about the enzymes responsible for the hydrolysis of neutral lipid esters, in particular of REs, in HSCs. In this study, we aimed to identify LD-associated neutral lipid hydrolases by a proteomic approach using the rat stellate cell line HSC-T6. First, we loaded cells with retinol and FAs to promote lipid synthesis and deposition within LDs. Then, LDs were isolated and lipid composition and the LD proteome were analyzed. Among other proteins, we found perilipin 2, adipose TG lipase (ATGL), and comparative gene identification-58 (CGI-58), known and established LD proteins. Bioinformatic search of the LD proteome for α/β-hydrolase fold-containing proteins revealed no yet uncharacterized neutral lipid hydrolases. In in vitro activity assays, we show that rat (r)ATGL, coactivated by rat (r)CGI-58, efficiently hydrolyzes TGs and REs. These findings suggest that rATGL and rCGI-58 are LD-resident proteins in HSCs and participate in the mobilization of both REs and TGs.     

The lipid droplet is an important organelle for hepatitis C virus production

The lipid droplet (LD) is an organelle that is used for the storage of neutral lipids. It dynamically moves through the cytoplasm, interacting with other organelles, including the endoplasmic reticulum (ER). These interactions are thought to facilitate the transport of lipids and proteins to other organelles. The hepatitis C virus (HCV) is a causative agent of chronic liver diseases. HCV capsid protein (Core) associates with the LD, envelope proteins E1 and E2 reside in the ER lumen, and the viral replicase is assumed to localize on ER-derived membranes. How and where HCV particles are assembled, however, is poorly understood. Here, we show that the LD is involved in the production of infectious virus particles. We demonstrate that Core recruits nonstructural (NS) proteins and replication complexes to LD-associated membranes, and that this recruitment is critical for producing infectious viruses. Furthermore, virus particles were observed in close proximity to LDs, indicating that some steps of virus assembly take place around LDs. This study reveals a novel function of LDs in the assembly of infectious HCV and provides a new perspective on how viruses usurp cellular functions.