ABA-INSENSITIVE 3 with or without FUSCA3 highly up-regulates lipid droplet proteins and activates oil accumulation.

ABA-INSENSITIVE 3 (ABI3) has long been known for activation of storage protein accumulation. A role of ABI3 on oil accumulation was previously suggested based on a decrease of oil content in seeds of abi3 mutant. However, this conclusion could not exclude possibilities of indirect or pleiotropic effects, such as through mutual regulatory interactions with FUSCA3 (FUS3), an activator of oil accumulation. To identify that ABI3 functions independent of the effects of related seed transcription factors, we expressed ABI3 under the control of an inducible promoter in tobacco BY2 cells and Arabidopsis rosette leaves. Inducible expression of ABI3 activated oil accumulation in these non-seed cells, demonstrating a general role of ABI3 in regulation of oil biosynthesis. Further expressing ABI3 in rosette leaves of fus3 knockout mutant still caused up to 3-fold greater triacylglycerol accumulation, indicating ABI3 can activate lipid accumulation independently of FUS3. Transcriptome analysis revealed that LIPID DROPLET PROTEIN (LDP) genes, including OLEOSINs and CALEOSINs, were up-regulated up to 1000-fold by ABI3 in the absence of FUS3, while the expression of WRINKLED1 was doubled. Taken together, our results provide genetic evidence that ABI3 activates oil accumulation with or without FUS3, most likely through up-regulating LDPs and WRINKLED1.

A look into Colorado potato beetle lipid metabolism through the lens of lipid storage droplet proteins.

The Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae) inflicts serious damage to potato plants by feeding ravenously on their leaves. Adult L.decemlineata have a photoperiod-induced dormancy response, also known as diapause, which allows them to survive severe winter conditions by digging into soil. Most insects that undergo diapause accumulate abundant lipid reserves prior to diapause and utilize most of them during the diapause. This process is likely to be governed by the interplay of lipid storage droplet proteins (LSDs), also known as perilipins, with the help of other proteins. Here, genes encoding L. decemlineata LSD1 and LSD2 were identified. Both were expressed primarily in the fat body with LdLSD1 and LdLSD2 being primarily expressed in adult and larval stages, respectively. LdLSD1 was up-regulated in starving larvae, while LdLSD2 was primarily expressed in feeding larvae. The expression pattern of LdLSD1 in adults during feeding, diapause and post-diapause contrasted to the total body fat levels, while the expression pattern of LdLSD2 was positively correlated with total body fat levels. RNA interference (RNAi) of LdLSD2 in larvae suggested a core role for LSD2 in the protection/assembly of storage lipids as this treatment reduced overall lipid droplet volume. These data shed light on the functions of these proteins in L. decemlineata and their roles in both diapause and during starvation.

Identification of Low-Abundance Lipid Droplet Proteins in Seeds and Seedlings.

The developmental program of seed formation, germination, and early seedling growth requires not only tight regulation of cell division and metabolism, but also concerted control of the structure and function of organelles, which relies on specific changes in their protein composition. Of particular interest is the switch from heterotrophic to photoautotrophic seedling growth, for which cytoplasmic lipid droplets (LDs) play a critical role as depots for energy-rich storage lipids. Here, we present the results of a bottom-up proteomics study analyzing the total protein fractions and LD-enriched fractions in eight different developmental phases during silique (seed) development, seed germination, and seedling establishment in Arabidopsis (Arabidopsis thaliana). The quantitative analysis of the LD proteome using LD-enrichment factors led to the identification of six previously unidentified and comparably low-abundance LD proteins, each of which was confirmed by intracellular localization studies with fluorescent protein fusions. In addition to these advances in LD protein discovery and the potential insights provided to as yet unexplored aspects in plant LD functions, our data set allowed for a comparative analysis of the LD protein composition throughout the various developmental phases examined. Among the most notable of the alterations in the LD proteome were those during seedling establishment, indicating a switch in the physiological function(s) of LDs after greening of the cotyledons. This work highlights LDs as dynamic organelles with functions beyond lipid storage.

Arabidopsis LDIP protein locates at a confined area within the lipid droplet surface and favors lipid droplet formation.

Lipid droplets (LDs) are cell organelles specialized in neutral lipid storage. Extendedly studied in seeds, LDs also accumulate in leaves during senescence or in response to abiotic stresses. However the mechanisms underlying their biogenesis remain relatively unknown. Here, we deciphered the distinct roles of two proteins during LD biogenesis: LD-associated protein 1 (AtLDAP1) and LDAP-interacting protein (AtLDIP). We demonstrated that AtLDIP overexpression favors the neo-formation of small LDs under growing conditions where LD accumulation is usually not observed. In addition, atldip knock-out mutant displayed fewer but larger LDs, confirming a role of AtLDIP in LD biogenesis. Interestingly, a synergistic effect of the overexpression of both AtLDIP and AtLDAP1 was observed, resulting in an increase of LD cluster occurrence and LD abundance within the clusters and the cells. AtLDIP overexpression has no significant impact on triacylglycerol and steryl ester accumulation but AtLDIP inactivation is associated with an increase of neutral lipid content, that is probably a consequence of the enlarged but less abundant LDs present in this line. Our localization study demonstrated that AtLDIP is localized at specific dotted sites within the LD in contrast to AtLDAP1 that covers the whole LD. In addition, AtLDIP sometimes localized away from the LD marker, but always associated with the ER network, suggesting a location at LD nascent sites within the ER. Taken together, our results suggested that AtLDIP promotes the formation of new LDs from ER localized TAG lenses.

PUX10 Is a Lipid Droplet-Localized Scaffold Protein That Interacts with CELL DIVISION CYCLE48 and Is Involved in the Degradation of Lipid Droplet Proteins.

The number of known proteins associated with plant lipid droplets (LDs) is small compared with other organelles. Many aspects of LD biosynthesis and degradation are unknown, and identifying and characterizing candidate LD proteins could help elucidate these processes. Here, we analyzed the proteome of LD-enriched fractions isolated from tobacco (Nicotiana tabacum) pollen tubes. Proteins that were highly enriched in comparison with the total or cytosolic fraction were further tested for LD localization via transient expression in pollen tubes. One of these proteins, PLANT UBX DOMAIN-CONTAINING PROTEIN10 (PUX10), is a member of the plant UBX domain-containing (PUX) protein family. This protein localizes to LDs via a unique hydrophobic polypeptide sequence and can recruit the AAA-type ATPase CELL DIVISION CYCLE48 (CDC48) protein via its UBX domain. PUX10 is conserved in Arabidopsis thaliana and expressed in embryos, pollen tubes, and seedlings. In pux10 knockout mutants in Arabidopsis, LD size is significantly increased. Proteomic analysis of pux10 mutants revealed a delayed degradation of known LD proteins, some of which possessed ubiquitination sites. We propose that PUX10 is involved in a protein degradation pathway at LDs, mediating an interaction between polyubiquitinated proteins targeted for degradation and downstream effectors such as CDC48.

Engineering lipid droplet assembly mechanisms for improved triacylglycerol accumulation in Saccharomyces cerevisiae.

Production of triacylglycerols (TAGs) through microbial fermentation is an emerging alternative to plant and animal-derived sources. The yeast Saccharomyces cerevisiae is a preferred organism for industrial use but has natively a very poor capacity of TAG production and storage. Here, we engineered S. cerevisiae for accumulation of high TAG levels through the use of structural and physiological factors that influence assembly and biogenesis of lipid droplets. First, human and fungal perilipin genes were expressed, increasing TAG content by up to 36% when expressing the human perilipin gene PLIN3. Secondly, expression of the FIT2 homologue YFT2 resulted in a 26% increase in TAG content. Lastly, the genes ERD1 and PMR1 were deleted in order to induce an endoplasmic reticulum stress response and stimulate lipid droplet formation, increasing TAG content by 72% for Δerd1. These new approaches were implemented in previously engineered strains that carry high flux of fatty acid biosynthesis and conversion of acyl-CoA into TAGs, resulting in improvements of up to 138% over those high-producing strains without any substantial growth effects or abnormal cell morphology. We find that these approaches not only represent a significant improvement of S. cerevisiae for TAG production, but also highlight the importance of lipid droplet dynamics for high lipid accumulation in yeast.

Bacterial lipid droplets bind to DNA via an intermediary protein that enhances survival under stress.

Lipid droplets (LDs) are multi-functional organelles consisting of a neutral lipid core surrounded by a phospholipid monolayer, and exist in organisms ranging from bacteria to humans. Here we study the functions of LDs in the oleaginous bacterium Rhodococcus jostii. We show that these LDs bind to genomic DNA through the major LD protein, MLDS, which increases survival rate of the bacterial cells under nutritional and genotoxic stress. MLDS expression is regulated by a transcriptional regulator, MLDSR, that binds to the operator and promoter of the operon encoding both proteins. LDs sequester MLDSR, controlling its availability for transcriptional regulation. Our findings support the idea that bacterial LDs can regulate nucleic acid function and facilitate bacterial survival under stress.

Synthetic Protein Scaffolds for Biosynthetic Pathway Colocalization on Lipid Droplet Membranes.

Eukaryotic biochemistry is organized throughout the cell in and on membrane-bound organelles. When engineering metabolic pathways this organization is often lost, resulting in flux imbalance and a loss of kinetic advantages from enzyme colocalization and substrate channeling. Here, we develop a protein-based scaffold for colocalizing multienzyme pathways on the membranes of intracellular lipid droplets. Scaffolds based on the plant lipid droplet protein oleosin and cohesin-dockerin interaction pairs recruited upstream enzymes in yeast ester biosynthesis to the native localization of the terminal reaction step, alcohol-O-acetyltransferase (Atf1). The native localization is necessary for high activity and pathway assembly in close proximity to Atf1 increased pathway flux. Screening a library of scaffold variants further showed that pathway structure can alter catalysis and revealed an optimized scaffold and pathway expression levels that produced ethyl acetate at a rate nearly 2-fold greater than unstructured pathways. This strategy should prove useful in spatially organizing other metabolic pathways with key lipid droplet-localized and membrane-bound reaction steps.

Function of lipid droplet-organelle interactions in lipid homeostasis.

Storage of non-polar lipids in ubiquitous eukaryotic organelles, lipid droplets (LDs), prevents the toxic consequences of unesterified fatty acids and provides a lipid reservoir that can be promptly used to satisfy cellular needs under multiple metabolic and physiological conditions. Tight temporal and spatial control of LD biogenesis and mobilization of neutral lipids is essential for the correct channelling of lipid intermediates to their various cellular destinations and the maintenance of cellular homeostasis. These functions are mediated by multiple interactions between LDs and other intracellular organelles that are required for the delivery of stored lipids. Here we review recent advances in the interactions of LDs with the endoplasmic reticulum (ER), mitochondria and vacuole/lysosome. This article is part of a Special Issue entitled: Membrane Contact Sites edited by Christian Ungermann and Benoit Kornmann.

Lipid droplet-associated proteins in atherosclerosis (Review).

Accumulation of atherosclerotic plaques in arterial walls leads to major cardiovascular diseases and stroke. Macrophages/foam cells are central components of atherosclerotic plaques, which populate the arterial wall in order to remove harmful modified low­density lipoprotein (LDL) particles, resulting in the accumulation of lipids, mostly LDL­derived cholesterol ester, in cytosolic lipid droplets (LDs). At present, LDs are recognized as dynamic organelles that govern cellular metabolic processes. LDs consist of an inner core of neutral lipids surrounded by a monolayer of phospholipids and free cholesterol, and contain LD­associated proteins (LDAPs) that regulate LD functions. Foam cells are characterized by an aberrant accumulation of cytosolic LDs, and are considered a hallmark of atherosclerotic lesions through all stages of development. Previous studies have investigated the mechanisms underlying foam cell formation, aiming to discover therapeutic strategies that target foam cells and intervene against atherosclerosis. It is well established that LDAPs have a major role in the pathogenesis of metabolic diseases caused by dysfunction of lipid metabolism, and several studies have linked LDAPs to the development of atherosclerosis. In this review, several foam cell­targeting pathways have been described, with an emphasis on the role of LDAPs in cholesterol mobilization from macrophages. In addition, the potential of LDAPs as therapeutic targets to prevent the progression and/or facilitate the regression of the disease has been discussed.

Lipid Droplet-Associated Proteins (LDAPs) Are Required for the Dynamic Regulation of Neutral Lipid Compartmentation in Plant Cells.

Eukaryotic cells compartmentalize neutral lipids into organelles called lipid droplets (LDs), and while much is known about the role of LDs in storing triacylglycerols in seeds, their biogenesis and function in nonseed tissues are poorly understood. Recently, we identified a class of plant-specific, lipid droplet-associated proteins (LDAPs) that are abundant components of LDs in nonseed cell types. Here, we characterized the three LDAPs in Arabidopsis (Arabidopsis thaliana) to gain insight to their targeting, assembly, and influence on LD function and dynamics. While all three LDAPs targeted specifically to the LD surface, truncation analysis of LDAP3 revealed that essentially the entire protein was required for LD localization. The association of LDAP3 with LDs was detergent sensitive, but the protein bound with similar affinity to synthetic liposomes of various phospholipid compositions, suggesting that other factors contributed to targeting specificity. Investigation of LD dynamics in leaves revealed that LD abundance was modulated during the diurnal cycle, and characterization of LDAP misexpression mutants indicated that all three LDAPs were important for this process. LD abundance was increased significantly during abiotic stress, and characterization of mutant lines revealed that LDAP1 and LDAP3 were required for the proper induction of LDs during heat and cold temperature stress, respectively. Furthermore, LDAP1 was required for proper neutral lipid compartmentalization and triacylglycerol degradation during postgerminative growth. Taken together, these studies reveal that LDAPs are required for the maintenance and regulation of LDs in plant cells and perform nonredundant functions in various physiological contexts, including stress response and postgerminative growth.