The lipodystrophy protein seipin is found at endoplasmic reticulum lipid droplet junctions and is important for droplet morphology

Abstract

Lipodystrophy is a disorder characterized by a loss of adipose tissue often accompanied by severe hypertriglyceridemia, insulin resistance, diabetes, and fatty liver. It can be inherited or acquired. The most severe inherited form is Berardinelli-Seip Congenital Lipodystrophy Type 2, associated with mutations in the BSCL2 gene. BSCL2 encodes seipin, the function of which has been entirely unknown. We now report the identification of yeast BSCL2/seipin through a screen to detect genes important for lipid droplet morphology. The absence of yeast seipin results in irregular lipid droplets often clustered alongside proliferated endoplasmic reticulum (ER); giant lipid droplets are also seen. Many small irregular lipid droplets are also apparent in fibroblasts from a BSCL2 patient. Human seipin can functionally replace yeast seipin, but a missense mutation in human seipin that causes lipodystrophy, or corresponding mutations in the yeast gene, render them unable to complement. Yeast seipin is localized in the ER, where it forms puncta. Almost all lipid droplets appear to be on the ER, and seipin is found at these junctions. Therefore, we hypothesize that seipin is important for droplet maintenance and perhaps assembly. In addition to detecting seipin, the screen identified 58 other genes whose deletions cause aberrant lipid droplets, including 2 genes encoding proteins known to activate lipin, a lipodystrophy locus in mice, and 16 other genes that are involved in endosomal-lysosomal trafficking. The genes identified in our screen should be of value in understanding the pathway of lipid droplet biogenesis and maintenance and the cause of some lipodystrophies.

Fig. 1.

Examples of strains with aberrant lipid body morphology. Cells were harvested in log or stationary phase, and lipid droplets were stained with BODIPY. (Scale bar, 2 μm.)

Fig. 2.

YLR404w is yeast BSCL2/seipin. (A) Alignment of seipins by using PROMALS (19). NCBI gene identification numbers (gi numbers) are shown after the sequences. Regions are highlighted yellow to indicate hydrophobic patches and conserved small amino acids are highlighted gray. Predicted secondary structure (PSIPRED) (29) is shown below the sequences. Predicted transmembrane α-helices are colored red. (B and C), Complementation of lipid droplet phenotype in ylr404wΔ by human and yeast seipins. Wild-type or the seipin-deleted strain was transformed with empty plasmid (pRS315) or plasmid-containing sequences encoding the long or short form of human seipin (HSeipinL or HSeipinS), A212P human seipin, yeast seipin (YSeipin), or yeast seipin with the mutations S224P or G225P. BODIPY-stained images are shown in B (scale bar, 5 μm); the histogram in C shows the number of lipid droplets per cell.

Fig. 3.

Abnormal lipid droplets in seipin-deficient cells. (A) ultrastructure of wild-type or the seipin KO yeast strains. Cells were cultured in glucose or oleate as indicated. Lipid droplets are in clusters in the KO (arrow in b, and in higher resolution in d–f). In oleate medium, lipid droplets in wild type are of uniform size (g) but are irregular in size, shape, and number in the KO strain (h and i). (All scale bars, 200 nm.) ER, endoplasmic reticulum; LB, lipid droplet; N, nucleus; V, vacuole. (B) Lipid droplets are aberrant in seipin-deficient human fibroblasts. Cells were stained with Hoechst and either Oil Red O (with or without culturing with oleate) or antibodies against the lipid droplet marker protein ADRP (without oleate). (Scale bars, 10 μm.) (C) Erg6p is targeted to the aberrant clusters (arrows), suggesting normal protein targeting. The merge image also incorporates brightfield to outline the cells. (Scale bar, 5 μm.) (D) The ER is concentrated in the aberrant clusters (arrows). Arrowhead indicates a giant lipid droplet. Note that lipid droplets appear to be attached to ER in the wild-type strain. (Scale bar = 5 μm.) (E) Proliferation of small lipid droplets in seipin-deficient fibroblasts, grown without oleate. Note the presence of many small clear organelles in the mutant cells. (Scale bars, 2 μm.)

Fig. 4.

Yeast seipin localizes in the ER and marks sites of contact with lipid droplets. (A) Seipin, tagged at either terminus with mCherry, localizes to the ER. Both seipin and GFP-HDEL were expressed on plasmids and driven by the PGK promoter. (B) Most lipid droplets appear bound to the ER. Droplets that were chromosomally tagged with Erg6p-mCherry also expressed the GFP-HDEL ER marker. (C) Seipin marks docking sites of lipid bodies. Seipin-mCherry, or inactive seipin-G225P-mCherry was chromosomally expressed at the seipin locus. CFP-HDEL, driven by the PGK promoter, was plasmid expressed. Lipid droplets were stained with BODIPY. Yellow boxes frame two cells showing clear seipin droplet proximity. Droplet clusters in cells expressing the mutant protein (null background) also colocalize seipin. (Scale bars, 5 μm.)