ER membrane phospholipids and surface tension control cellular lipid droplet formation

Cells convert excess energy into neutral lipids that are made in the endoplasmic reticulum (ER) bilayer. The lipids are then packaged into spherical or budded lipid droplets (LDs) covered by a phospholipid monolayer containing proteins. LDs play a key role in cellular energy metabolism and homeostasis. A key unanswered question in the life of LDs is how they bud off from the ER. Here, we tackle this question by studying the budding of artificial LDs from model membranes. We find that the bilayer phospholipid composition and surface tension are key parameters of LD budding. Phospholipids have differential LD budding aptitudes, and those inducing budding decrease the bilayer tension. We observe that decreasing tension favors the egress of neutral lipids from the bilayer and LD budding. In cells, budding conditions favor the formation of small LDs. Our discovery reveals the importance of altering ER physical chemistry for controlled cellular LD formation

Interaction of hepatitis C virus core protein with lipid droplets : mechanism and function

Une étape majeure pour le maintien de l'état d'infection par le Virus de l'Hépatite C dans les cellules est la liaison de la protéine Core de la capside à la membrane des gouttelettes lipidiques formées dans le foie. Core se lie avec une hélice amphipathique à l’interface eau-huile des gouttelettes lipidiques. Le mécanisme de ce passage n'a pas encore été élucidé et la régulation du lien reste floue. Comprendre ce trafic intracellulaire nécessite, entre autres, une bonne connaissance de la biophysique des interactions protéine-membrane, en particulier des interfaces d'émulsion. Peu a été fait dans ce sens. Pour ce projet, nous étudions le mécanisme du trafic cellulaire de Core et ses partenaires entre le réticulum endoplasmique et les gouttelettes lipidiques. Nous adoptons une approche multidisciplinaire. Pour surmonter les complexités associées aux multiples interactions de Core et qui empêchent actuellement de comprendre la liaison de la protéine, nous avons reconstitué sa liaison sur des membranes modèles. Nous formons des gouttes d'émulsions huile dans eau imitant les gouttelettes lipidiques et des vésicules imitant le réticulum. Nous déterminons ainsi les conditions favorisant la liaison de Core sur la GL. Cette approche, associée à des expériences in vivo, est innovante et apporte une compréhension qui fait actuellement défaut.A major step for the maintenance of the hepatitis C infection state in the cells is the binding of the Core protein from the capsid to the lipid droplets membrane formed in the liver. Core binds with an amphipathic helix to use it from the bilayer membrane of the endoplasmic reticulum to the water-oil interface of the lipid droplets. The mechanism of this passage has not yet been elucidated and the regulation of the link remains unclear. Understanding this physical Core traffic requires, among other things, a good knowledge of the biophysics of protein-membrane interaction, especially on emulsion interfaces. Little has been done in this direction. For this project, we investigated the mechanism of cellular traffic of Core and its partners between endoplasmic reticulum and lipid droplets. We adopted a multidisciplinary approach. To overcome the complexities associated with the multiple interactions of Core, and which currently prevent an understanding of the binding of the protein, we reconstituted it binding on model membranes. We formed drops of oil-in-water emulsions, mimicking the lipid droplets, and vesicles mimicking the endoplasmic reticulum. We thus determined the conditions favoring the binding of Core on the lipid droplets. This approach, coupled with in vivo manipulations, is innovative and bring an understanding that is currently lacking

Interaction de la protéine core du virus de l’hépatite C avec les corps lipidiques : mécanisme et fonction

A major step for the maintenance of the hepatitis C infection state in the cells is the binding of the Core protein from the capsid to the lipid droplets membrane formed in the liver. Core binds with an amphipathic helix to use it from the bilayer membrane of the endoplasmic reticulum to the water-oil interface of the lipid droplets. The mechanism of this passage has not yet been elucidated and the regulation of the link remains unclear. Understanding this physical Core traffic requires, among other things, a good knowledge of the biophysics of protein-membrane interaction, especially on emulsion interfaces. Little has been done in this direction. For this project, we investigated the mechanism of cellular traffic of Core and its partners between endoplasmic reticulum and lipid droplets. We adopted a multidisciplinary approach. To overcome the complexities associated with the multiple interactions of Core, and which currently prevent an understanding of the binding of the protein, we reconstituted it binding on model membranes. We formed drops of oil-in-water emulsions, mimicking the lipid droplets, and vesicles mimicking the endoplasmic reticulum. We thus determined the conditions favoring the binding of Core on the lipid droplets. This approach, coupled with in vivo manipulations, is innovative and bring an understanding that is currently lacking.Une étape majeure pour le maintien de l'état d'infection par le Virus de l'Hépatite C dans les cellules est la liaison de la protéine Core de la capside à la membrane des gouttelettes lipidiques formées dans le foie. Core se lie avec une hélice amphipathique à l’interface eau-huile des gouttelettes lipidiques. Le mécanisme de ce passage n'a pas encore été élucidé et la régulation du lien reste floue. Comprendre ce trafic intracellulaire nécessite, entre autres, une bonne connaissance de la biophysique des interactions protéine-membrane, en particulier des interfaces d'émulsion. Peu a été fait dans ce sens. Pour ce projet, nous étudions le mécanisme du trafic cellulaire de Core et ses partenaires entre le réticulum endoplasmique et les gouttelettes lipidiques. Nous adoptons une approche multidisciplinaire. Pour surmonter les complexités associées aux multiples interactions de Core et qui empêchent actuellement de comprendre la liaison de la protéine, nous avons reconstitué sa liaison sur des membranes modèles. Nous formons des gouttes d'émulsions huile dans eau imitant les gouttelettes lipidiques et des vésicules imitant le réticulum. Nous déterminons ainsi les conditions favorisant la liaison de Core sur la GL. Cette approche, associée à des expériences in vivo, est innovante et apporte une compréhension qui fait actuellement défaut

N-terminal domain of PB1-F2 protein of influenza A virus can fold into amyloid-like oligomers and damage cholesterol and cardiolipid containing membranes

PB1-F2 protein is a factor of virulence of influenza A viruses which increases the mortality and morbidity associated with infection. Most seasonal H1N1 Influenza A viruses express nowadays a truncated version of PB1-F2. Here we show that truncation of PB1-F2 modified supramolecular organization of the protein in a membrane-mimicking environment. In addition, full-length PB1-F2(1–90) and C-terminal PB1-F2 domain (53–90), efficiently permeabilized various anionic liposomes while N-terminal domain PB1-F2(1–52) only lysed cholesterol and cardiolipin containing lipid bilayers. These findings suggest that the truncation of PB1-F2 may impact the pathogenicity of a given virus strain.Accepted versio

HCV Core protein needs triacylglycerols to fold onto the endoplasmic reticulum membrane

International audienceLipid droplets (LDs) are involved in viral infections, but exactly how remains unclear. Here, we study the hepatitis C virus (HCV) whose Core capsid protein binds to LDs but is also involved in the assembly of virions at the endoplasmic reticulum (ER) bilayer. We found that the amphipathic helix-containing domain of Core, D2, senses triglycerides (TGs) rather than LDs per se. In the absence of LDs, D2 can bind to the ER membrane but only if TG molecules are present in the bilayer. Accordingly, the pharmacological inhibition of the diacylglycerol O-acyltransferase enzymes, mediating TG synthesis in the ER, inhibits D2 association with the bilayer. We found that TG molecules enable D2 to fold into alpha helices. Sequence analysis reveals that D2 resembles the apoE lipid-binding region. Our data support that TG in LDs promotes the folding of Core, which subsequently relocalizes to contiguous ER regions. During this motion, Core may carry TG molecules to these regions where HCV lipoviroparticles likely assemble. Consistent with this model, the inhibition of Arf1/COPI, which decreases LD surface accessibility to proteins and ER-LD material exchange, severely impedes the assembly of virions. Altogether, our data uncover a critical function of TG in the folding of Core and HCV replication and reveals, more broadly, how TG accumulation in the ER may provoke the binding of soluble amphipathic helix-containing proteins to the ER bilayer

Membrane Asymmetry Imposes Directionality on Lipid Droplet Emergence from the ER

International audienceDuring energy bursts, neutral lipids fabricated within the ER bilayer demix to form lipid droplets (LDs). LDs bud off mainly in the cytosol where they regulate metabolism and multiple biological processes. They indeed become accessible to most enzymes and can interact with other organelles. How such directional emergence is achieved remains elusive. Here, we found that this directionality is controlled by an asymmetry in monolayer surface coverage. Model LDs emerge on the membrane leaflet of higher coverage, which is improved by the insertion of proteins and phospholipids. In cells, continuous LD emergence on the cytosol would require a constant refill of phospholipids to the ER cytosolic leaflet. Consistent with this model, cells deficient in phospholipids present an increased number of LDs exposed to the ER lumen and compensate by remodeling ER shape. Our results reveal an active cooperation between phospholipids and proteins to extract LDs from ER

Dual binding motifs underpin the hierarchical association of perilipins1-3 with lipid droplets.

Lipid droplets (LDs) in all eukaryotic cells are coated with at least one of the perilipin family of proteins. They all regulate key intracellular lipases but do so to significantly different extents. Where more than one perilipin is expressed in a cell, they associate with LDs in a hierarchical manner. In vivo, this means that lipid flux control in a particular cell or tissue type is heavily influenced by the specific perilipins present on its LDs. Despite their early discovery, exactly how perilipins target LDs and why they displace each other in a ‘hierarchical’ manner remains unclear. They all share an amino-terminal 11-mer repeat amphipathic region suggested to be involved in LD targeting. Here, we show that in vivo this domain functions as a primary highly reversible LD targeting motif in perilipins1-3 and, in vitro, we document reversible and competitive binding between a wildtype purified perilipin1 11-mer repeat peptide and a mutant with reduced binding affinity to both ‘naked’ and phospholipid coated oil-water interfaces. We also present data suggesting that a second carboxy-terminal 4-helix bundle domain stabilizes LD binding in perilipin1 more effectively than in perilipin2, whereas in perilipin3 it weakens binding. These findings suggest that dual amphipathic helical regions mediat

Dual binding motifs underpin the hierarchical association of perilipins1-3 with lipid droplets.

Lipid droplets (LDs) in all eukaryotic cells are coated with at least one of the perilipin (Plin) family of proteins. They all regulate key intracellular lipases but do so to significantly different extents. Where more than one Plin is expressed in a cell, they associate with LDs in a hierarchical manner. In vivo, this means that lipid flux control in a particular cell or tissue type is heavily influenced by the specific Plins present on its LDs. Despite their early discovery, exactly how Plins target LDs and why they displace each other in a "hierarchical" manner remains unclear. They all share an amino-terminal 11-mer repeat (11mr) amphipathic region suggested to be involved in LD targeting. Here, we show that, in vivo, this domain functions as a primary highly reversible LD targeting motif in Plin1-3, and, in vitro, we document reversible and competitive binding between a wild-type purified Plin1 11mr peptide and a mutant with reduced binding affinity to both "naked" and phospholipid-coated oil-water interfaces. We also present data suggesting that a second carboxy-terminal 4-helix bundle domain stabilizes LD binding in Plin1 more effectively than in Plin2, whereas it weakens binding in Plin3. These findings suggest that dual amphipathic helical regions mediate LD targeting and underpin the hierarchical binding of Plin1-3 to LDs

Structural insights into perilipin 3 membrane association in response to diacylglycerol accumulation

Abstract Lipid droplets (LDs) are dynamic organelles that contain an oil core mainly composed of triglycerides (TAG) that is surrounded by a phospholipid monolayer and LD-associated proteins called perilipins (PLINs). During LD biogenesis, perilipin 3 (PLIN3) is recruited to nascent LDs as they emerge from the endoplasmic reticulum. Here, we analyze how lipid composition affects PLIN3 recruitment to membrane bilayers and LDs, and the structural changes that occur upon membrane binding. We find that the TAG precursors phosphatidic acid and diacylglycerol (DAG) recruit PLIN3 to membrane bilayers and define an expanded Perilipin-ADRP-Tip47 (PAT) domain that preferentially binds DAG-enriched membranes. Membrane binding induces a disorder to order transition of alpha helices within the PAT domain and 11-mer repeats, with intramolecular distance measurements consistent with the expanded PAT domain adopting a folded but dynamic structure upon membrane binding. In cells, PLIN3 is recruited to DAG-enriched ER membranes, and this requires both the PAT domain and 11-mer repeats. This provides molecular details of PLIN3 recruitment to nascent LDs and identifies a function of the PAT domain of PLIN3 in DAG binding