Adipogenin Dictates Adipose Tissue Expansion by Facilitating the Assembly of a Dodecameric Seipin Complex.

Adipogenin (Adig) is an evolutionarily conserved microprotein and is highly expressed in adipose tissues and testis. Here, we identify Adig as a critical regulator for lipid droplet formation in adipocytes. We determine that Adig interacts directly with seipin, leading to the formation of a rigid complex. We solve the structure of the seipin/Adig complex by Cryo-EM at 2.98Å overall resolution. Surprisingly, seipin can form two unique oligomers, undecamers and dodecamers. Adig selectively binds to the dodecameric seipin complex. We further find that Adig promotes seipin assembly by stabilizing and bridging adjacent seipin subunits. Functionally, Adig plays a key role in generating lipid droplets in adipocytes. In mice, inducible overexpression of Adig in adipocytes substantially increases fat mass, with enlarged lipid droplets. It also elevates thermogenesis during cold exposure. In contrast, inducible adipocyte-specific Adig knockout mice manifest aberrant lipid droplet formation in brown adipose tissues and impaired cold tolerance.

Fatty Acyl Coenzyme A Synthetase Fat1p Regulates Vacuolar Structure and Stationary-Phase Lipophagy in Saccharomyces cerevisiae.

During yeast stationary phase, a single spherical vacuole (lysosome) is created by the fusion of several small ones. Moreover, the vacuolar membrane is reconstructed into two distinct microdomains. Little is known, however, about how cells maintain vacuolar shape or regulate their microdomains. Here, we show that Fat1p, a fatty acyl coenzyme A (acyl-CoA) synthetase and fatty acid transporter, and not the synthetases Faa1p and Faa4p, is essential for vacuolar shape preservation, the development of vacuolar microdomains, and cell survival in stationary phase of the yeast Saccharomyces cerevisiae. Furthermore, Fat1p negatively regulates general autophagy in both log- and stationary-phase cells. In contrast, Fat1p promotes lipophagy, as the absence of FAT1 limits the entry of lipid droplets into the vacuole and reduces the degradation of liquid droplet (LD) surface proteins. Notably, supplementing with unsaturated fatty acids or overexpressing the desaturase Ole1p can reverse all aberrant phenotypes caused by FAT1 deficiency. We propose that Fat1p regulates stationary phase vacuolar morphology, microdomain differentiation, general autophagy, and lipophagy by controlling the degree of fatty acid saturation in membrane lipids. IMPORTANCE The ability to sense environmental changes and adjust the levels of cellular metabolism is critical for cell viability. Autophagy is a recycling process that makes the most of already-existing energy resources, and the vacuole/lysosome is the ultimate autophagic processing site in cells. Lipophagy is an autophagic process to select degrading lipid droplets. In yeast cells in stationary phase, vacuoles fuse and remodel their membranes to create a single spherical vacuole with two distinct membrane microdomains, which are required for yeast lipophagy. In this study, we discovered that Fat1p was capable of rapidly responding to changes in nutritional status and preserving cell survival by regulating membrane lipid saturation to maintain proper vacuolar morphology and the level of lipophagy in the yeast S. cerevisiae. Our findings shed light on how cells maintain vacuolar structure and promote the differentiation of vacuole surface microdomains for stationary-phase lipophagy.

Seipin forms a flexible cage at lipid droplet formation sites.

Lipid droplets (LDs) form in the endoplasmic reticulum by phase separation of neutral lipids. This process is facilitated by the seipin protein complex, which consists of a ring of seipin monomers, with a yet unclear function. Here, we report a structure of S. cerevisiae seipin based on cryogenic-electron microscopy and structural modeling data. Seipin forms a decameric, cage-like structure with the lumenal domains forming a stable ring at the cage floor and transmembrane segments forming the cage sides and top. The transmembrane segments interact with adjacent monomers in two distinct, alternating conformations. These conformations result from changes in switch regions, located between the lumenal domains and the transmembrane segments, that are required for seipin function. Our data indicate a model for LD formation in which a closed seipin cage enables triacylglycerol phase separation and subsequently switches to an open conformation to allow LD growth and budding.

Seipin: harvesting fat and keeping adipocytes healthy.

Seipin is a key protein in the assembly of cytoplasmic lipid droplets (cLDs) and their maintenance at endoplasmic reticulum (ER)-LD junctions; the absence of seipin results in generalized lipodystrophy. How seipin mediates LD dynamics and prevents lipodystrophy are not well understood. New evidence suggests that seipin attracts triglyceride monomers from the ER to sites of droplet formation. By contrast, seipin may not be directly involved in the assembly of nuclear LDs and may actually suppress their formation at a distance. Seipin promotes adipogenesis, but lipodystrophy may also involve postadipogenic effects. We hypothesize that among these are a cycle of runaway lipolysis and lipotoxicity caused by aberrant LDs, resulting in a depletion of fat stores and a failure of adipose and other cells to thrive.

Building the lipid droplet assembly complex.

In this issue, Choudhary et al. (2020. J. Cell Biol.https://doi.org/10.1083/jcb.201910177) address the nature of the ER subdomain from which lipid droplets emanate and how several assembly proteins interact. Their data indicate that seipin/Nem1 marks these sites and provide a detailed working model for assembling the protein complex.

Spatial compartmentalization of lipid droplet biogenesis.

Lipid droplets (LDs) are ubiquitous organelles that store metabolic energy in the form of neutral lipids (typically triacylglycerols and steryl esters). Beyond being inert energy storage compartments, LDs are dynamic organelles that participate in numerous essential metabolic functions. Cells generate LDs de novo from distinct sub-regions at the endoplasmic reticulum (ER), but what determines sites of LD formation remains a key unanswered question. Here, we review the factors that determine LD formation at the ER, and discuss how they work together to spatially and temporally coordinate LD biogenesis. These factors include lipid synthesis enzymes, assembly proteins, and membrane structural requirements. LDs also make contact with other organelles, and these inter-organelle contacts contribute to defining sites of LD production. Finally, we highlight emerging non-canonical roles for LDs in maintaining cellular homeostasis during stress.

LDAF1 Holds the Key to Seipin Function.

Seipin is an ER protein important for the assembly of cytoplasmic lipid droplets. In this issue of Developmental Cell, Chung et al. (2019) show that a stable seipin-binding protein, LDAF1/promethin, functions with seipin by attracting triacylglycerol and then allowing this lipid to accumulate and partition into nascent droplets.

The assembly of lipid droplets and their roles in challenged cells.

Cytoplasmic lipid droplets are important organelles in nearly every eukaryotic and some prokaryotic cells. Storing and providing energy is their main function, but they do not work in isolation. They respond to stimuli initiated either on the cell surface or in the cytoplasm as conditions change. Cellular stresses such as starvation and invasion are internal insults that evoke changes in droplet metabolism and dynamics. This review will first outline lipid droplet assembly and then discuss how droplets respond to stress and in particular nutrient starvation. Finally, the role of droplets in viral and microbial invasion will be presented, where an unresolved issue is whether changes in droplet abundance promote the invader, defend the host, to try to do both. The challenges of stress and infection are often accompanied by changes in physical contacts between droplets and other organelles. How these changes may result in improving cellular physiology, an ongoing focus in the field, is discussed.

Understanding the Lipid Droplet Proteome and Protein Targeting.

Reporting in this issue of Developmental Cell, Bersuker et al. (2018) adapt APEX technology to lipid droplets for a more accurate view of the droplet proteome and Prévost et al. (2018) provide important insights into the basis of droplet protein targeting that altogether extend the understanding of this organelle.

Pet10p is a yeast perilipin that stabilizes lipid droplets and promotes their assembly.

Pet10p is a yeast lipid droplet protein of unknown function. We show that it binds specifically to and is stabilized by droplets containing triacylglycerol (TG). Droplets isolated from cells with a PET10 deletion strongly aggregate, appear fragile, and fuse in vivo when cells are cultured in oleic acid. Pet10p binds early to nascent droplets, and their rate of appearance is decreased in pet10Δ Moreover, Pet10p functionally interacts with the endoplasmic reticulum droplet assembly factors seipin and Fit2 to maintain proper droplet morphology. The activity of Dga1p, a diacylglycerol acyltransferase, and TG accumulation were both 30-35% lower in the absence of Pet10p. Pet10p contains a PAT domain, a defining property of perilipins, which was not previously known to exist in yeast. We propose that the core functions of Pet10p and other perilipins extend beyond protection from lipases and include the preservation of droplet integrity as well as collaboration with seipin and Fit2 in droplet assembly and maintenance.

The collaborative work of droplet assembly.

Three proteins have been implicated in the assembly of cytoplasmic lipid droplets: seipin, FIT2, and perilipin. This review examines the current theories of seipin function as well as the evidence for the involvement of all three proteins in droplet biogenesis, and ends with a proposal of how they collaborate to regulate the formation of droplets. This article is part of a Special Issue entitled: Recent Advances in Lipid Droplet Biology edited by Rosalind Coleman and Matthijs Hesselink.

Dissecting seipin function: the localized accumulation of phosphatidic acid at ER/LD junctions in the absence of seipin is suppressed by Sei1p(ΔNterm) only in combination with Ldb16p.

BACKGROUND: Seipin is required for the correct assembly of cytoplasmic lipid droplets. In the absence of the yeast seipin homolog Sei1p (formerly Fld1p), droplets are slow to bud from the endoplasmic reticulum, lack the normal component of proteins on their surface, are highly heterogeneous in size and shape, often bud into the nucleus, and promote local proliferation of the endoplasmic reticulum in which they become tangled. But the mechanism by which seipin catalyzes lipid droplet formation is still uncertain. RESULTS: Seipin prevents a localized accumulation of phosphatidic acid (PA puncta) at ER-droplet junctions. PA puncta were detected with three different probes: Opi1p, Spo20p(51-91) and Pah1p. A system of droplet induction was used to show that PA puncta were not present until droplets were formed; the puncta appeared regardless of whether droplets consisted of triacylglycerol or steryl ester. Deletion strains were used to demonstrate that a single phosphatidic acid-producing enzyme is not responsible for the generation of the puncta, and the puncta remain resistant to overexpression of enzymes that metabolize phosphatidic acid, suggesting that this lipid is trapped in a latent compartment. Suppression of PA puncta requires the first 14 amino acids of Sei1p (Nterm), a domain that is also important for initiation of droplet assembly. Consistent with recent evidence that Ldb16p and Sei1p form a functional unit, the PA puncta phenotype in the ldb16Δ sei1Δ strain was rescued by human seipin. Moreover, PA puncta in the sei1Δ strain expressing Sei1p(ΔNterm) was suppressed by overexpression of Ldb16p, suggesting a functional interaction of Nterm with this protein. Overexpression of both Sei1p and Ldb16p, but not Sei1p alone, is sufficient to cause a large increase in droplet number. However, Ldb16p alone increases triacylglycerol accumulation in the ldb16Δ sei1Δ background. CONCLUSION: We hypothesize that seipin prevents formation of membranes with extreme curvature at endoplasmic reticulum/droplet junctions that would attract phosphatidic acid. While Ldb16p alone can affect triacylglycerol accumulation, proper droplet formation requires the collaboration of Sei1p and Ldb16.

Arabidopsis SEIPIN Proteins Modulate Triacylglycerol Accumulation and Influence Lipid Droplet Proliferation.

The lipodystrophy protein SEIPIN is important for lipid droplet (LD) biogenesis in human and yeast cells. In contrast with the single SEIPIN genes in humans and yeast, there are three SEIPIN homologs in Arabidopsis thaliana, designated SEIPIN1, SEIPIN2, and SEIPIN3. Essentially nothing is known about the functions of SEIPIN homologs in plants. Here, a yeast (Saccharomyces cerevisiae) SEIPIN deletion mutant strain and a plant (Nicotiana benthamiana) transient expression system were used to test the ability of Arabidopsis SEIPINs to influence LD morphology. In both species, expression of SEIPIN1 promoted accumulation of large-sized lipid droplets, while expression of SEIPIN2 and especially SEIPIN3 promoted small LDs. Arabidopsis SEIPINs increased triacylglycerol levels and altered composition. In tobacco, endoplasmic reticulum (ER)-localized SEIPINs reorganized the normal, reticulated ER structure into discrete ER domains that colocalized with LDs. N-terminal deletions and swapping experiments of SEIPIN1 and 3 revealed that this region of SEIPIN determines LD size. Ectopic overexpression of SEIPIN1 in Arabidopsis resulted in increased numbers of large LDs in leaves, as well as in seeds, and increased seed oil content by up to 10% over wild-type seeds. By contrast, RNAi suppression of SEIPIN1 resulted in smaller seeds and, as a consequence, a reduction in the amount of oil per seed compared with the wild type. Overall, our results indicate that Arabidopsis SEIPINs are part of a conserved LD biogenesis machinery in eukaryotes and that in plants these proteins may have evolved specialized roles in the storage of neutral lipids by differentially modulating the number and sizes of lipid droplets.

The lipid droplet-a well-connected organelle.

Our knowledge of inter-organellar communication has grown exponentially in recent years. This review focuses on the interactions that cytoplasmic lipid droplets have with other organelles. Twenty-five years ago droplets were considered simply particles of coalesced fat. Ten years ago there were hints from proteomics studies that droplets might interact with other structures to share lipids and proteins. Now it is clear that the droplets interact with many if not most cellular structures to maintain cellular homeostasis and to buffer against insults such as starvation. The evidence for this statement, as well as probes to understand the nature and results of droplet interactions, are presented.

The life cycle of lipid droplets.

Proteomic studies have revealed many potential functions of cytoplasmic lipid droplets, and recent activity has confirmed that these bona fide organelles are central not only for lipid storage and metabolism, but for development, immunity, and pathogenesis by several microbes. There has been a burst of recent activity on the assembly, maintenance and turnover of lipid droplets that reveals fresh insights. This review summarizes several novel findings in initiation of lipid droplet assembly, protein targeting, droplet fusion, and turnover of droplets through lipophagy.

Seipin performs dissectible functions in promoting lipid droplet biogenesis and regulating droplet morphology.

Seipin is necessary for both adipogenesis and lipid droplet (LD) organization in nonadipose tissues; however, its molecular function is incompletely understood. Phenotypes in the seipin-null mutant of Saccharomyces cerevisiae include aberrant droplet morphology (endoplasmic reticulum-droplet clusters and size heterogeneity) and sensitivity of droplet size to changes in phospholipid synthesis. It has not been clear, however, whether seipin acts in initiation of droplet synthesis or at a later step. Here we utilize a system of de novo droplet formation to show that the absence of seipin results in a delay in droplet appearance with concomitant accumulation of neutral lipid in membranes. We also demonstrate that seipin is required for vectorial budding of droplets toward the cytoplasm. Furthermore, we find that the normal rate of droplet initiation depends on 14 amino acids at the amino terminus of seipin, deletion of which results in fewer, larger droplets that are consistent with a delay in initiation but are otherwise normal in morphology. Importantly, other functions of seipin, namely vectorial budding and resistance to inositol, are retained in this mutant. We conclude that seipin has dissectible roles in both promoting early LD initiation and in regulating LD morphology, supporting its importance in LD biogenesis.

Seipin: from human disease to molecular mechanism.

The most-severe form of congenital generalized lipodystrophy (CGL) is caused by mutations in BSCL2/seipin. Seipin is a homo-oligomeric integral membrane protein in the endoplasmic reticulum that concentrates at junctions with cytoplasmic lipid droplets (LDs). While null mutations in seipin are responsible for lipodystrophy, dominant mutations cause peripheral neuropathy and other nervous system pathologies. We first review the clinical aspects of CGL and the discovery of the responsible genetic loci. The structure of seipin, its normal isoforms, and mutations found in patients are then presented. While the function of seipin is not clear, seipin gene manipulation in yeast, flies, mice, and human cells has recently yielded a trove of information that suggests roles in lipid metabolism and LD assembly and maintenance. A model is presented that attempts to bridge these new data to understand the role of this fascinating protein.