RESUMO
Pathogen infection induces massive reprogramming of host primary metabolism. Lipid and fatty acid (FA) metabolism is generally disrupted by pathogens and co-opted for their proliferation. Lipid droplets (LDs) that play important roles in regulating cellular lipid metabolism are utilized by a variety of pathogens in mammalian cells. However, the function of LDs during pathogenic infection in plants remains unknown. We show here that infection by rice black streaked dwarf virus (RBSDV) affects the lipid metabolism of maize, which causes elevated accumulation of C18 polyunsaturated fatty acids (PUFAs) leading to viral proliferation and symptom development. The overexpression of one of the two novel LD-associated proteins (LDAPs) of maize (ZmLDAP1 and ZmLDAP2) induces LD clustering. The core capsid protein P8 of RBSDV interacts with ZmLDAP2 and prevents its degradation through the ubiquitin-proteasome system mediated by a UBX domain-containing protein, PUX10. In addition, silencing of ZmLDAP2 downregulates the expression of FA desaturase genes in maize, leading to a decrease in C18 PUFAs levels and suppression of RBSDV accumulation. Our findings reveal that plant virus may recruit LDAP to regulate cellular FA metabolism to promote viral multiplication and infection. These results expand the knowledge of LD functions and viral infection mechanisms in plants.
Assuntos
Ácidos Graxos , Doenças das Plantas , Proteínas de Plantas , Replicação Viral , Zea mays , Zea mays/virologia , Zea mays/metabolismo , Zea mays/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Doenças das Plantas/virologia , Ácidos Graxos/metabolismo , Metabolismo dos Lipídeos , Proteínas Associadas a Gotículas Lipídicas/metabolismo , Proteínas Associadas a Gotículas Lipídicas/genética , Gotículas Lipídicas/metabolismo , Gotículas Lipídicas/virologia , Vírus de Plantas/fisiologia , Regulação da Expressão Gênica de Plantas , Reoviridae/fisiologiaRESUMO
Lipid droplet (LD) function relies on proteins partitioning between the endoplasmic reticulum (ER) phospholipid bilayer and the LD monolayer membrane to control cellular adaptation to metabolic changes. It has been proposed that these hairpin proteins integrate into both membranes in a similar monotopic topology, enabling their passive lateral diffusion during LD emergence at the ER. Here, we combine biochemical solvent-accessibility assays, electron paramagnetic resonance spectroscopy and intra-molecular crosslinking experiments with molecular dynamics simulations, and determine distinct intramembrane positionings of the ER/LD protein UBXD8 in ER bilayer and LD monolayer membranes. UBXD8 is deeply inserted into the ER bilayer with a V-shaped topology and adopts an open-shallow conformation in the LD monolayer. Major structural rearrangements are required to enable ER-to-LD partitioning. Free energy calculations suggest that such structural transition is unlikely spontaneous, indicating that ER-to-LD protein partitioning relies on more complex mechanisms than anticipated and providing regulatory means for this trans-organelle protein trafficking.
Assuntos
Retículo Endoplasmático , Gotículas Lipídicas , Simulação de Dinâmica Molecular , Retículo Endoplasmático/metabolismo , Gotículas Lipídicas/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Bicamadas Lipídicas/metabolismo , Bicamadas Lipídicas/química , Transporte Proteico , Animais , Proteínas Associadas a Gotículas Lipídicas/metabolismo , Proteínas Associadas a Gotículas Lipídicas/química , Proteínas Associadas a Gotículas Lipídicas/genéticaRESUMO
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.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Proteínas Associadas a Gotículas Lipídicas/genética , Proteínas Associadas a Gotículas Lipídicas/metabolismo , Sementes/metabolismo , Fatores de Transcrição/genéticaRESUMO
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.
Assuntos
Besouros , Proteínas Associadas a Gotículas Lipídicas , Metabolismo dos Lipídeos , Animais , Besouros/genética , Besouros/metabolismo , Besouros/fisiologia , Diapausa/fisiologia , Corpo Adiposo/metabolismo , Regulação da Expressão Gênica , Genes de Insetos , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Larva/genética , Larva/metabolismo , Proteínas Associadas a Gotículas Lipídicas/genética , Proteínas Associadas a Gotículas Lipídicas/metabolismo , Interferência de RNA , Inanição/metabolismoRESUMO
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.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas Associadas a Gotículas Lipídicas/genética , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos/genética , Nicotiana/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Clonagem Molecular , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Expressão Gênica , Técnicas de Inativação de Genes , Proteínas Associadas a Gotículas Lipídicas/metabolismo , Gotículas Lipídicas/química , Folhas de Planta/genética , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sementes/genética , Sementes/metabolismo , Nicotiana/metabolismo , Triglicerídeos/biossínteseRESUMO
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.
Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas Associadas a Gotículas Lipídicas/metabolismo , Plântula/metabolismo , Sementes/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Germinação/genética , Germinação/fisiologia , Proteínas Associadas a Gotículas Lipídicas/genética , Proteoma/genética , Proteoma/metabolismo , Plântula/genética , Sementes/genéticaRESUMO
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.
Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Associadas a Gotículas Lipídicas/metabolismo , Gotículas Lipídicas/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Ciclo Celular/genética , Proteínas Associadas a Gotículas Lipídicas/genética , Poliubiquitina/metabolismo , Proteômica/métodosRESUMO
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.
Assuntos
Microbiologia Industrial/métodos , Gotículas Lipídicas/metabolismo , Engenharia Metabólica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Triglicerídeos/metabolismo , Estresse do Retículo Endoplasmático/genética , Humanos , Proteínas Associadas a Gotículas Lipídicas/genética , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Triglicerídeos/biossínteseRESUMO
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.
Assuntos
Proteínas de Bactérias/metabolismo , DNA Bacteriano/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas Associadas a Gotículas Lipídicas/metabolismo , Gotículas Lipídicas/metabolismo , Rhodococcus/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Sítios de Ligação , DNA Bacteriano/genética , Proteínas Associadas a Gotículas Lipídicas/genética , Gotículas Lipídicas/química , Gotículas Lipídicas/efeitos dos fármacos , Gotículas Lipídicas/ultraestrutura , Metabolismo dos Lipídeos/efeitos dos fármacos , Viabilidade Microbiana , Nitrogênio/deficiência , Nitrogênio/farmacologia , Óperon , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Rhodococcus/efeitos dos fármacos , Rhodococcus/genética , Rhodococcus/ultraestrutura , Estresse Fisiológico , Transcrição GênicaRESUMO
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.
Assuntos
Proteínas Associadas a Gotículas Lipídicas/genética , Gotículas Lipídicas/metabolismo , Engenharia Metabólica/métodos , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/fisiologia , Melhoramento Genético/métodos , Proteínas Associadas a Gotículas Lipídicas/metabolismo , Frações Subcelulares , Biologia Sintética/métodosRESUMO
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.
Assuntos
Homeostase , Membranas Intracelulares/metabolismo , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos , Animais , Humanos , Proteínas Associadas a Gotículas Lipídicas/genética , Proteínas Associadas a Gotículas Lipídicas/metabolismoRESUMO
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.
Assuntos
Aterosclerose/etiologia , Aterosclerose/metabolismo , Proteínas Associadas a Gotículas Lipídicas/metabolismo , Animais , Aterosclerose/patologia , Transporte Biológico , Colesterol/metabolismo , Ésteres do Colesterol/metabolismo , Modelos Animais de Doenças , Células Espumosas/metabolismo , Células Espumosas/patologia , Humanos , Hidrólise , Proteínas Associadas a Gotículas Lipídicas/genética , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos , Lipoproteínas LDL/metabolismo , Macrófagos/metabolismo , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patologiaRESUMO
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.