The seipin complex Fld1/Ldb16 stabilizes ER–lipid droplet contact sites

Lipid droplets (LDs) are storage organelles consisting of a neutral lipid core surrounded by a phospholipid monolayer and a set of LD-specific proteins. Most LD components are synthesized in the endoplasmic reticulum (ER), an organelle that is often physically connected with LDs. How LD identity is established while maintaining biochemical and physical connections with the ER is not known. Here, we show that the yeast seipin Fld1, in complex with the ER membrane protein Ldb16, prevents equilibration of ER and LD surface components by stabilizing the contact sites between the two organelles. In the absence of the Fld1/Ldb16 complex, assembly of LDs results in phospholipid packing defects leading to aberrant distribution of lipid-binding proteins and abnormal LDs. We propose that the Fld1/Ldb16 complex facilitates the establishment of LD identity by acting as a diffusion barrier at the ER–LD contact sites.     

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.     

Regulated localization of Rab18 to lipid droplets: effects of lipolytic stimulation and inhibition of lipid droplet catabolism

Rab GTPases are crucial regulators of membrane traffic. Here we have examined a possible association of Rab proteins with lipid droplets (LDs), neutral lipid-containing organelles surrounded by a phospholipid monolayer, also known as lipid bodies, which have been traditionally considered relatively inert storage organelles. Although we found close apposition between LDs and endosomal compartments labeled by expressed Rab5, Rab7, or Rab11 constructs, there was no detectable labeling of the LD surface itself by these Rab proteins. In contrast, GFP-Rab18 localized to LDs and immunoelectron microscopy showed direct association with the monolayer surface. Green fluorescent protein (GFP)-Rab18-labeled LDs underwent oscillatory movements in a localized area as well as sporadic, rapid, saltatory movements both in the periphery of the cell and toward the perinuclear region. In both adipocytes and non-adipocyte cell lines Rab18 localized to a subset of LDs. To gain insights into this specific localization, Rab18 was co-expressed with Cav3DGV, a truncation mutant of caveolin-3 shown to inhibit the catabolism and motility of lipid droplets. GFP-Rab18 and mRFP-Cav3DGV labeled mutually exclusive subpopulations of LDs. Moreover, in 3T3-L1 adipocytes, stimulation of lipolysis increased the localization of Rab18 to LDs, an effect reversed by beta-adrenergic antagonists. These results show that a Rab protein localizes directly to the monolayer surface of LDs. In addition, association with the LD surface was increased following stimulation of lipolysis and inhibited by a caveolin mutant suggesting that recruitment of Rab18 is regulated by the metabolic state of individual LDs.     

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.     

Identification of the major functional proteins of prokaryotic lipid droplets

Storage of cellular triacylglycerols (TAGs) in lipid droplets (LDs) has been linked to the progression of many metabolic diseases in humans, and to the development of biofuels from plants and microorganisms. However, the biogenesis and dynamics of LDs are poorly understood. Compared with other organisms, bacteria seem to be a better model system for studying LD biology, because they are relatively simple and are highly efficient in converting biomass to TAG. We obtained highly purified LDs from Rhodococcus sp. RHA1, a bacterium that can produce TAG from many carbon sources, and then comprehensively characterized the LD proteome. Of the 228 LD-associated proteins identified, two major proteins, ro02104 and PspA, constituted about 15% of the total LD protein. The structure predicted for ro02104 resembles that of apolipoproteins, the structural proteins of plasma lipoproteins in mammals. Deletion of ro02104 resulted in the formation of supersized LDs, indicating that ro02104 plays a critical role in cellular LD dynamics. The putative α helix of the ro02104 LD-targeting domain (amino acids 83-146) is also similar to that of apolipoproteins. We report the identification of 228 proteins in the proteome of prokaryotic LDs, identify a putative structural protein of this organelle, and suggest that apolipoproteins may have an evolutionarily conserved role in the storage and trafficking of neutral lipids.     

Quantitative electron microscopy shows uniform incorporation of triglycerides into existing lipid droplets

The lipid droplet (LD) is an organelle with a lipid ester core and a surface phospholipid monolayer. The mechanism of LD biogenesis is not well understood. The present study aimed to elucidate the LD growth process, for which we developed a new electron microscopic method that quantifies the proportion of existing and newly synthesized triglycerides in individual LDs. Our method takes advantage of the reactivity of unsaturated fatty acids and osmium tetroxide, which imparts LDs an electron density that reflects fatty acid composition. With this method, existing triglyceride-rich LDs in 3Y1 fibroblasts were observed to incorporate newly synthesized triglycerides at a highly uniform rate. This uniformity and its persistence even after microtubules were depolymerized suggest that triglycerides in fibroblasts are synthesized in the local vicinity of individual LDs and then incorporated. In contrast, LDs in 3T3-L1 adipocytes showed heterogeneity in the rate at which lipid esters were incorporated, indicating different mechanisms of LD growth in fibroblasts and adipocytes.     

Adding fuel to the fire: The lipid droplet and its associated proteins in cancer progression

A lipid droplet (LD) is an organelle that consists of a phospholipid monolayer and a neutral lipid core, with proteins embedded in or attached to its surface. Until recently, cancers had long been regarded as genetic disorders with the abnormal activation of oncogenes and inactivation of tumor suppressor genes before their quality of a metabolic disorder began to be recognized. The last decade has witnessed the recognition of several metabolic characteristics of cancer cells, among which one is the accumulation of lipid droplets; therefore, attention has been given to exploring the role of LDs in carcinomas. In addition, there has been a remarkable expansion in understanding the complexity of LD''s function in cellular homeostasis, including but not limited to energy supply, endoplasmic reticulum (ER) stress and oxidative stress management, or lipotoxicity alleviation. Thus, lipid droplet-associated proteins, which to a great extent determine the dynamics of a lipid droplet, have attracted the interest of numerous cancer researchers and their potential as cancer diagnostic biomarkers and therapeutic targets has been affirmed by emerging evidence. In this review, we systematically summarize the critical role of LDs in cancer and then focus on four categories of lipid droplet-associated proteins having the most direct influence on LD biosynthesis (diacylglycerol acyltransferase 1 (DGAT1) and diacylglycerol acyltransferase 2 (DGAT2)), degradation (adipose triglyceride lipase (ATGL)), and two renowned protein families on the LD surface (perilipins and cell death-inducing DNA fragmentation factor alpha-like effectors (CIDEs)). In this way, we aim to highlight their important role in tumor progression and their potential in clinical applications.     

High confidence proteomic analysis of yeast LDs identifies additional droplet proteins and reveals connections to dolichol synthesis and sterol acetylation

Accurate protein inventories are essential for understanding an organelle’s functions. The lipid droplet (LD) is a ubiquitous intracellular organelle with major functions in lipid storage and metabolism. LDs differ from other organelles because they are bounded by a surface monolayer, presenting unique features for protein targeting to LDs. Many proteins of varied functions have been found in purified LD fractions by proteomics. While these studies have become increasingly sensitive, it is often unclear which of the identified proteins are specific to LDs. Here we used protein correlation profiling to identify 35 proteins that specifically enrich with LD fractions of Saccharomyces cerevisiae. Of these candidates, 30 fluorophore-tagged proteins localize to LDs by microscopy, including six proteins, several with human orthologs linked to diseases, which we newly identify as LD proteins (Cab5, Rer2, Say1, Tsc10, YKL047W, and YPR147C). Two of these proteins, Say1, a sterol deacetylase, and Rer2, a cis-isoprenyl transferase, are enzymes involved in sterol and polyprenol metabolism, respectively, and we show their activities are present in LD fractions. Our results provide a highly specific list of yeast LD proteins and reveal that the vast majority of these proteins are involved in lipid metabolism.     

Lipid droplet proteins and metabolic diseases

Lipid droplets (LDs) are ubiquitous cellular organelles for lipid storage which are composed of a neutral lipid core bounded by a protein decorated phospholipid monolayer. Although lipid storage is their most obvious function, LDs are far from inert as they participate in maintaining lipid homeostasis through lipid synthesis, metabolism, and transportation. Furthermore, they are involved in cell signaling and other molecular events closely associated with human disease such as dyslipidemia, obesity, lipodystrophy, diabetes, fatty liver, atherosclerosis, and others. The last decade has seen a great increase in the attention paid to LD biology. Regardless, many fundamental features of LD biology remain obscure. In this review, we will discuss key aspects of LD biology including their biogenesis, growth and regression. We will also summarize the current knowledge about the role LDs play in human disease, especially from the perspective of the dynamics of the associated proteins. This article is part of a Special issue entitled Cardiac adaptations to obesity, diabetes and insulin resistance, edited by Professors Jan F.C. Glatz, Jason R.B. Dyck and Christine Des Rosiers.     

Mammalian lipid droplets: structural,pathological, immunological and anti-toxicological roles

Mammalian lipid droplets (LDs) are specialized cytosolic organelles consisting of a neutral lipid core surrounded by a membrane made up of a phospholipid monolayer and a specific population of proteins that varies according to the location and function of each LD. Over the past decade, there have been significant advances in the understanding of LD biogenesis and functions. LDs are now recognized as dynamic organelles that participate in many aspects of cellular homeostasis plus other vital functions. LD biogenesis is a complex, highly-regulated process with assembly occurring on the endoplasmic reticulum although aspects of the underpinning molecular mechanisms remain elusive. For example, it is unclear how many enzymes participate in the biosynthesis of the neutral lipid components of LDs and how this process is coordinated in response to different metabolic cues to promote or suppress LD formation and turnover. In addition to enzymes involved in the biosynthesis of neutral lipids, various scaffolding proteins play roles in coordinating LD formation. Despite their lack of ultrastructural diversity, LDs in different mammalian cell types are involved in a wide range of biological functions. These include roles in membrane homeostasis, regulation of hypoxia, neoplastic inflammatory responses, cellular oxidative status, lipid peroxidation, and protection against potentially toxic intracellular fatty acids and lipophilic xenobiotics. Herein, the roles of mammalian LDs and their associated proteins are reviewed with a particular focus on their roles in pathological, immunological and anti-toxicological processes.     

ER-localized phosphatidylethanolamine synthase plays a conserved role in lipid droplet formation

The asymmetric distribution of phospholipids in membranes is a fundamental principle of cellular compartmentalization and organization. Phosphatidylethanolamine (PE), a nonbilayer phospholipid that contributes to organelle shape and function, is synthesized at several subcellular localizations via semiredundant pathways. Previously, we demonstrated in budding yeast that the PE synthase Psd1, which primarily operates on the mitochondrial inner membrane, is additionally targeted to the ER. While ER-localized Psd1 is required to support cellular growth in the absence of redundant pathways, its physiological function is unclear. We now demonstrate that ER-localized Psd1 sublocalizes on the ER to lipid droplet (LD) attachment sites and show it is specifically required for normal LD formation. We also find that the role of phosphatidylserine decarboxylase (PSD) enzymes in LD formation is conserved in other organisms. Thus we have identified PSD enzymes as novel regulators of LDs and demonstrate that both mitochondria and LDs in yeast are organized and shaped by the spatial positioning of a single PE synthesis enzyme.     

Born this way – Biogenesis of lipid droplets from specialized ER subdomains

Both the endoplasmic reticulum (ER) and lipid droplets (LDs) are key players in lipid handling. In addition to this functional connection, the two organelles are also tightly linked due to the fact that the ER is the birthplace of LDs. LDs have an atypical architecture, consisting of a neutral lipid core that is covered by a phospholipid monolayer. LD biogenesis starts with neutral lipid synthesis in the ER membrane and formation of small neutral lipid lenses between its leaflets, followed by budding of mature LDs toward the cytosol.Several ER proteins have been identified that are required for efficient LD formation, among them seipin, Pex30, and FIT2. Recent evidence indicates that these LD biogenesis factors might cooperate with specific lipids, thus generating ER subdomains optimized for LD assembly. Intriguingly, LD biogenesis reacts dynamically to nutrient stress, resulting in a spatial reorganization of LD formation in the ER.     

The Proteome of Cholesteryl-Ester-Enriched Versus Triacylglycerol-Enriched Lipid Droplets

Within cells, lipids are stored in the form of lipid droplets (LDs), consisting of a neutral lipid core, surrounded by a phospholipid monolayer and an outer layer of protein. LDs typically accumulate either triacylglycerol (TAG) and diacylglycerol or cholesteryl ester (CE), depending on the type of tissue. Recently, there has been an increased interest in the proteins that surround LDs. LD proteins have been found to be quite diverse, from structural proteins to metabolic enzymes, proteins involved in vesicular transport, and proteins that may play a role in LD formation. Previous proteomics analyses have focused on TAG-enriched LDs, whereas CE-enriched LDs have been largely ignored. Our study has compared the LD proteins from CE-enriched LDs to TAG-enriched LDs in steroidogenic cells. In primary rat granulosa cells loaded with either HDL to produce CE-enriched LDs or fatty acids to produce TAG-enriched LDs, 61 proteins were found to be elevated in CE-enriched LDs and 40 proteins elevated in TAG-enriched LDs with 278 proteins in similar amounts. Protein expression was further validated by selected reaction monitoring (SRM) mass spectrometry (MS). SRM verified expression of 25 of 27 peptides that were previously detected by tandem mass tagging MS. Several proteins were confirmed to be elevated in CE-enriched LDs by SRM including the intermediate filament vimentin. This study is the first to compare the proteins found on CE-enriched LDs with TAG-enriched LDs and constitutes the first step in creating a better understanding of the proteins found on CE-enriched LDs in steroidogenic cells.     

DFCP1 associates with lipid droplets

Double FYVE‐containing protein 1 (DFCP1) is ubiquitously expressed, participates in intracellular membrane trafficking and labels omegasomes through specific interactions with phosphatidylinositol‐3‐phosphate (PI3P). Previous studies showed that subcellular DFCP1 proteins display multi‐organelle localization, including in the endoplasmic reticulum (ER), Golgi apparatus and mitochondria. However, its localization and function on lipid droplets (LDs) remain unclear. Here, we demonstrate that DFCP1 localizes to the LD upon oleic acid incubation. The ER‐targeted domain of DFCP1 is indispensable for its LD localization, which is further enhanced by double FYVE domains. Inhibition of PI3P binding at the FYVE domain through wortmannin treatment or double mutation at C654S and C770S have no effect on DFCP1's LD localization. These show that the mechanisms for DFCP1 targeting the omegasome and LDs are different. DFCP1 deficiency in MEF cells causes an increase in LD number and reduces LD size. Interestingly, DFCP1 interacts with GTP‐bound Rab18, an LD‐associated protein. Taken together, our work demonstrates the dynamic localization of DFCP1 is regulated by nutritional status in response to cellular metabolism.     

Proteome of skeletal muscle lipid droplet reveals association with mitochondria and apolipoprotein a-I

The lipid droplet (LD) is a universal organelle governing the storage and turnover of neutral lipids. Mounting evidence indicates that elevated intramuscular triglyceride (IMTG) in skeletal muscle LDs is closely associated with insulin resistance and Type 2 Diabetes Mellitus (T2DM). Therefore, the identification of the skeletal muscle LD proteome will provide some clues to dissect the mechanism connecting IMTG with T2DM. In the present work, we identified 324 LD-associated proteins in mouse skeletal muscle LDs through mass spectrometry analysis. Besides lipid metabolism and membrane traffic proteins, a remarkable number of mitochondrial proteins were observed in the skeletal muscle LD proteome. Furthermore, imaging by fluorescence microscopy and transmission electronic microscopy (TEM) directly demonstrated that mitochondria closely adhere to LDs in vivo. Moreover, our results revealed for the first time that apolipoprotein A-I (apo A-I), the principal apolipoprotein of high density lipoprotein (HDL) particles, was also localized on skeletal muscle LDs. Further studies verified that apo A-I was expressed endogenously by skeletal muscle cells. In conclusion, we report the protein composition and characterization of skeletal muscle LDs and describe a novel LD-associated protein, apo A-I.     

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.     

Recent Advances in Understanding Proteins Involved in Lipid Droplet Formation,Growth and Fusion

Lipid droplets （LDs） were once viewed as simple, inert lipid micelles. However, they are now known to be organelles with a rich proteome involved in a myriad of cellular processes. LDs are heterogeneous in nature with different sizes and compositions of phospholipids, neutral lipids and proteins. This review takes a focused look at the roles of proteins involved in the regulation of LD formation, expansion, and morphology. The related proteins are summarized such as the fat-specific protein （Fsp27）, fat storage-inducing trans- membrane （FIT） proteins, seipin and ADP-ribosylation factor 1-coat protein complex I （Arf-COPI）. Finally, we present important challenges in LD biology for a deeper understanding of this dynamic organelle to be achieved.     

Lipid droplets in skeletal muscle during grass snake (Natrix natrix L.) development

Lipid droplets (LDs) are common organelles observed in Eucaryota. They are multifunctional organelles (involved in lipid storage, metabolism, and trafficking) that originate from endoplasmic reticulum (ER). LDs consist of a neutral lipid core, made up of diacyl- and triacylglycerols (DAGs and TAGs) and cholesterol esters (CEs), surrounded by a phospholipid monolayer and proteins, which are necessary for their structure and dynamics.Here, we report the protein and lipid composition as well as characterization and dynamics of grass snake (Natrix natrix) skeletal muscle LDs at different developmental stages. In the present study, we used detailed morphometric, LC-MS, quantitative lipidomic analyses of LDs isolated from the skeletal muscles of the snake embryos, immunofluorescence, and TEM.Our study also provides a valuable insight concerning the LDs' multifunctionality and ability to interact with a variety of organelles. These LD features are reflected in their proteome composition, which contains scaffold proteins, metabolic enzymes signalling polypeptides, proteins necessary for the formation of docking sites, and many others. We also provide insights into the biogenesis and growth of muscle LDs goes beyond the conventional mechanism based on the synthesis and incorporation of TAGs and LD fusion. We assume that the formation and functioning of grass snake muscle LDs are based on additional mechanisms that have not yet been identified, which could be related to the unique features of reptiles that are manifested in the after-hatching period of life, such as a reptile-specific strategy for energy saving during hibernation.     

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.