Seipin Is a Discrete Homooligomer

Abstract

Seipin is a transmembrane protein that resides in the endoplasmic reticulum and concentrates at junctions between the ER and cytosolic lipid droplets. Mutations in the human seipin gene, including the missense mutation A212P, lead to congenital generalized lipodystrophy (CGL), characterized by the lack of normal adipose tissue and accumulation of fat in liver and muscles. In both yeast and CGL patient fibroblasts, seipin is required for normal lipid droplet morphology; in its absence droplets appear to bud abnormally from the ER. Here we report the first purification and physical characterization of seipin. Yeast seipin is in a large discrete protein complex. Affinity purification demonstrated that seipin is the main if not exclusive protein in the complex. Detergent sucrose gradients in H2O, and D2O and gel filtration were used to determine the size of the seipin complex and account for detergent binding. Both seipin-myc13 (seipin fused to 13 tandem copies of the myc epitope) expressed from the endogenous promoter and overexpressed seipin-mCherry form ~500 kDa proteins consisting of about 9 copies of seipin. The yeast orthologue of the human A212P allele forms only smaller complexes and is unstable; we hypothesize that this accounts for its null phenotype in humans. Seipin appears as a toroid by negative staining electron microscopy. We speculate that seipin plays at least a structural role in organizing droplets or in communication between droplets and ER.

Lipin 2 Binds Phosphatidic Acid by the Electrostatic Hydrogen Bond Switch Mechanism Independent of Phosphorylation

“…We have previously shown that within cells the lipins form tetramers (34), and by atomic force microscopy, we have found that recombinant lipin 1 can form even higher order structures on lipin bilayers (35). In addition, lipin 1 has been found to interact with the human lipodystrophy protein seipin (36), and seipin itself has been found to form large oligomeric structures (37)(38)(39). Although the identification of lipin localization to punctate spots is interesting, further study will be necessary to uncover the nature and/or function of lipin protein cellular localization to these structures.…”

A role for seipin in lipid droplet dynamics and inheritance in yeast

“…A potential candidate protein involved in mediating such an interaction in yeast is Fld1, the yeast orthologue of mammalian seipin, mutations of which are implicated in a severe inherited SeipBerardinelli congenital lipodystrophy (Fei et al, 2008b;Szymanski et al, 2007). Yeast seipin is an oligomeric transmembrane protein that localizes to the interface between lipid droplets and the ER, and was suggested to play a role in organizing lipid droplets (Binns et al, 2010). Yeast mutants lacking Fld1 show a diverse pattern of lipid droplets in the cell population, differing in both size and subcellular distribution (Fei et al, 2008b;Szymanski et al, 2007).…”

“…A prime candidate protein involved in mediating contacts between lipid droplets and the ER membrane is Fld1, the yeast seipin, because its absence results in an abnormal lipid droplet morphology that is also dependent on the cultivation conditions (Binns et al, 2010;Fei et al, 2008b;Szymanski et al, 2007). In our experiments, cells were cultivated either in rich medium or in SC minimal medium to stationary phase, at which time point, differences in the distribution of lipid droplets in wild-type and fld1D mutant cells are more pronounced.…”

“…It was suggested that the generation of super-sized lipid droplets in the fld1 strain is caused by a change of the phospholipid composition of the membrane monolayer of the organelle (Fei et al, 2008b). By contrast, the occurrence of irregular, 'chaotic budding' of lipid droplets and the localization of Fld1 at lipid droplets to ER interfaces indicate a structural function of Fld1 in organizing lipid droplets or in the interaction between lipid droplets and the ER (Binns et al, 2010;Szymanski et al, 2007).…”

Seipin: from human disease to molecular mechanism

“…While physiological binding partners of seipin have yet to be identifi ed, it is clear that seipin can self-associate ( 51,59 ). When extracted from yeast membranes with Triton X-100, seipin behaves as a discrete and stable oligomer.…”