Membrane Asymmetry Imposes Directionality on Lipid Droplet Emergence from the ER

Chorlay, Aymeric; Monticelli, Luca; Ferreira, Joana Veríssimo; M’Barek, Kalthoum Ben; Ajjaji, Dalila; Wang, Sihui; Johnson, Errin; Beck, Rainer; Omrane, Mohyeddine; Beller, Mathias; Carvalho, Pedro; Thiam, Abdou Rachid

doi:10.1016/j.devcel.2019.05.003

Cited by 126 publications

(165 citation statements)

References 75 publications

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“…10g, h). Consistent with this proposition, artificial LD embedded into giant unilamellar vesicle emerge toward the side having greater coverage with proteins and phospholipids, which reduce surface tension of the monolayer (Chorlay et al, 2019). Continued synthesis of TAG at Fld1/Nem1 sites results in maturation of LDs, which remain associated with 22 the ER membrane at Fld1/Nem1 sites (Fig.…”

Section: Discussionsupporting

confidence: 65%

Seipin and Nem1 establish discrete ER subdomains to initiate yeast lipid droplet biogenesis

Choudhary

Atab

Mizzon

et al. 2020

Preprint

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Lipid droplets (LDs) are fat storage organelles that originate from the endoplasmic reticulum (ER).Relatively little is known about how sites of LD formation are selected, and which proteins/lipids are necessary for the process. Here, we show that LDs induced by the yeast triacylglycerol (TAG)synthases Lro1 and Dga1 are formed at discrete ER subdomains defined by seipin (Fld1), and a regulator of diacylglycerol (DAG) production, Nem1. Fld1 and Nem1 colocalize to ER-LD contact sites. We find that Fld1 and Nem1 localize to ER subdomains independently of each other and of LDs, but both are required for the subdomains to recruit the TAG synthases and additional LD biogeneiss factors: Yft2, Pex30, Pet10, and Erg6. These subdomains become enriched in DAG.We conclude that Fld1 and Nem1 are both necessary to recruit proteins to ER subdomains where LD biogenesis occurs.10-, 30-60-, 90-, 120-min ( Fig S4F), at 10-min, 2 h, 4 h, 21 h (Fig. 1C), and at 21 h (Fig. 1D, 4D, S1G). Cells were lysed using a Precellys 24 homogenizier, lipids were extracted as described and equal aliquots were brought to dryness. To quantify TAG and SE, lipids were spotted onto silica gel 60 thin layer chromatography (TLC) plates (Merck, Darmstadt, Germany) and developed with petroleum ether/diethylether/acetic acid (70:30:2; per vol).Glycerophospholipids were separated by TLC as described (Vaden et al., 2005). TLC plates were exposed to a tritium-sensitive screen, visualized and quantified using a phosphorimager (GE Healthcare). Brightness and contrast of the TIFF images were adjusted using Image J (NIH). Western blot analysisCells were grown over night in SC raffinose media, diluted to 1 OD600 ml in SC galactose media and cells corresponding to 3 OD600 units were harvested at 0-, 2-, 4-, 6-, 21-h time points.Samples were extracted by NaOH, and proteins were precipitated using trichloroacetic acid (TCA), and resuspended into sample buffer (Horvath and Riezman, 1994). Proteins were separated using SDS-PAGE and detected using a monoclonal antibody against GFP (Roche Diagnostics, Rotkreuz, Switzerland, dilution 1:2000), or Kar2 (Santa Cruz Biotechnology, Dallas, TX, dilution 1:5000) and using horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology, Dallas, TX, #2302, dilution 1:10000). Western blot experiments were performed at least twice with similar results. Quantification and statistical analysisUnless otherwise stated, data were obtained from at least three independent experiments.The images presented are representative of the results obtained. Quantitative results are expressed

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Section: Discussionsupporting

confidence: 65%

Seipin and Nem1 establish discrete ER subdomains to initiate yeast lipid droplet biogenesis

Choudhary

Atab

Mizzon

et al. 2020

Preprint

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“…9, steps g and h). Consistent with this proposition, artificial LDs embedded into giant unilamellar vesicles emerge toward the side having greater coverage with proteins and phospholipids, which reduces surface tension of the monolayer (Chorlay et al, 2019). Continued synthesis of TAG at Fld1/Nem1 sites results in maturation of LDs, which remain associated with the ER membrane at Fld1/Nem1 sites (Fig.…”

Section: Discussionsupporting

confidence: 63%

Seipin and Nem1 establish discrete ER subdomains to initiate yeast lipid droplet biogenesis

Choudhary

Atab

Mizzon

et al. 2020

Journal of Cell Biology

111

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Lipid droplets (LDs) are fat storage organelles that originate from the endoplasmic reticulum (ER). Relatively little is known about how sites of LD formation are selected and which proteins/lipids are necessary for the process. Here, we show that LDs induced by the yeast triacylglycerol (TAG)-synthases Lro1 and Dga1 are formed at discrete ER subdomains defined by seipin (Fld1), and a regulator of diacylglycerol (DAG) production, Nem1. Fld1 and Nem1 colocalize to ER–LD contact sites. We find that Fld1 and Nem1 localize to ER subdomains independently of each other and of LDs, but both are required for the subdomains to recruit the TAG-synthases and additional LD biogenesis factors: Yft2, Pex30, Pet10, and Erg6. These subdomains become enriched in DAG. We conclude that Fld1 and Nem1 are both necessary to recruit proteins to ER subdomains where LD biogenesis occurs.

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“…These results suggest that LDs may emerge from the ER bilayer with different interfacial energies with respect to their neutral lipid content. This interfacial energy could be further reduced by the recruitment of amphipathic molecules such as AHs (Chorlay et al, 2019). Thus, we hypothesized that AHs would be differently recruited to the surface of emerging aLDs made of different neutral lipids.…”

Section: Resultsmentioning

confidence: 99%

“…Most neutral lipids are fabricated at the ER bilayer and encapsulated within the bilayer leaflets. When their concentration reaches a threshold, they condense into spherical LDs, which further emerge in the cytosol (Chorlay et al, 2019; Henne et al, 2018; Thiam and Forêt, 2016; Walther et al, 2017). LDs are covered by a single phospholipid (PL) monolayer embedded with proteins, which determine most LD biological functions (Kory et al, 2016).…”

Section: Introductionmentioning

confidence: 99%

Neutral lipids regulate amphipathic helix affinity for model lipid droplets

Chorlay

Thiam

2020

Journal of Cell Biology

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Cellular lipid droplets (LDs) have a neutral lipid core shielded from the aqueous environment by a phospholipid monolayer containing proteins. These proteins define the biological functions of LDs, and most of them bear amphipathic helices (AH), which can selectively target to LDs, or to LD subsets. How such binding preference happens remains poorly understood. Here, we found that artificial LDs made of different neutral lipids but presenting equal phospholipid packing densities differentially recruit AHs. Varying the phospholipid density shifts the binding levels, but the differential recruitment is unchanged. We found that the binding level of AHs is defined by their interaction preference with neutral lipids and ability to decrease surface tension. The phospholipid packing level regulates mainly the amount of neutral lipid accessible. Therefore, it is the hydrophobic nature of the phospholipid packing voids that controls the binding level of AHs. Our data bring us a major step closer to understanding the binding selectivity of AHs to lipid membranes.

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Membrane Asymmetry Imposes Directionality on Lipid Droplet Emergence from the ER

Cited by 126 publications

References 75 publications

Seipin and Nem1 establish discrete ER subdomains to initiate yeast lipid droplet biogenesis

Seipin and Nem1 establish discrete ER subdomains to initiate yeast lipid droplet biogenesis

Seipin and Nem1 establish discrete ER subdomains to initiate yeast lipid droplet biogenesis

Neutral lipids regulate amphipathic helix affinity for model lipid droplets

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