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

BMC Cell Biology, Dec 2015

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.

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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

Han et al. BMC Cell Biology Dissecting seipin function: the localized accumulation of phosphatidic acid at ER/LD junctions in the absence of seipin is ΔNterm suppressed by Sei1p only in combination with Ldb16p Sungwon Han 0 Derk D. Binns 0 Yu-Fang Chang 1 Joel M. Goodman 0 0 Department of Pharmacology, University of Texas Southwestern Medical School , Dallas, TX 75390-9041 , USA 1 Department of Molecular Biology and Genetics, Cornell University , Ithaca, NY 14853-2703 , USA 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 Sei1pΔNterm 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. Seipin; Lipid droplets; Phosphatidic acid; FLD1; SEI1; LDB16 Background Cytoplasmic lipid droplets, found in most eukaryotic and some prokaryotic cells, store energy in the form of neutral lipids, particularly triacylglycerols (TAG) and steryl esters (SE), which are enwrapped by a single phospholipid monolayer and associated proteins [ 1 ]. They emanate from the endoplasmic reticulum (ER) and can remain attached there [ 2 ]; they also generate contacts with other organelles, presumably for the purpose of lipid exchange [ 3, 4 ]. The synthesis of droplets is becoming better understood. The general view of early steps is that collections of neutral lipids, as they accumulate between the ER leaflets after synthesis, eventually will assume a spherical shape, remaining covered with ER membrane as they bud outward [ 5, 6 ]. However, proteins may control this reaction [7]. For example, Fit2 is found in the ER, binds to neutral lipids in isolation, and clearly affects droplet formation in some systems [ 8, 9 ]. Our laboratory has been focusing on seipin, a protein first identified in severe congenital generalized lipodystrophy, in which adipose tissue fails to develop [10]. Seipin was also identified in yeast screens for morphologically aberrant lipid droplets [ 11, 12 ]. The absence of seipin in yeast leads to both irregularly shaped clusters of tiny droplets enwrapped in the ER, and “supersized” droplets; the two forms depends on levels of cell phospholipids, as the supersized phenotype is suppressed by high levels of the phospholipid precursor, inositol [13]. Droplets in seipin-null cells also frequently bud into the nucleus, a phenomenon rarely seen in wild type cells [ 14 ]. Moreover, they lack, or have diminished levels, of a subset of droplet proteins [ 15, 16 ]. The absence of seipin also results in slow formation of droplets under conditions where the amount of total cellular neutral lipid is identical. When droplets are produced in this system, they are often actually clusters of microdroplets with ER fragments attached, as if a product of bilayer membrane stability [ 14 ]. Overall, these studies suggest that seipin is important in controlling the initiation of droplets as well as affecting the size, topology and protein composition of the (...truncated)


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Sungwon Han, Derk Binns, Yu-Fang Chang, Joel Goodman. 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, BMC Cell Biology, 2015, pp. 29, 16, DOI: 10.1186/s12860-015-0075-3