Seipin is a discrete homooligomer.
Seipin is a discrete homooligomer.
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DOI：
10.1021/bi1013003
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发表时间：
2010-12-21
期刊：
影响因子：
2.9
通讯作者：
Goodman JM
中科院分区：
文献类型：
--
作者：
Binns D;Lee S;Hilton CL;Jiang QX;Goodman JM
Seipin是一种跨膜蛋白，存在于内质网中，并集中在ER和胞质脂滴之间的连接处。人seipin基因的突变，包括错义突变A212 P，导致先天性全身性脂肪营养不良（CGL），其特征是缺乏正常脂肪组织，脂肪在肝脏和肌肉中积累。在酵母和CGL患者成纤维细胞中，seipin是正常脂滴形态所必需的;在其不存在的情况下，液滴似乎从ER中异常出芽。在这里，我们报告的第一个纯化和物理特性的seipin。酵母Seipin是一个大的离散蛋白质复合物。亲和纯化表明，seipin是主要的，如果不是唯一的蛋白质在复合物中。洗涤剂蔗糖在H2O和D2 O中的梯度和凝胶过滤用于确定seipin复合物的大小并解释洗涤剂结合。从内源启动子表达的seipin-myc 13（seipin融合到myc表位的13个串联拷贝）和过表达的seipin-mCherry形成约500 kDa的蛋白质，其由约9个拷贝的seipin组成。人类A212 P等位基因的酵母直向同源物仅形成较小的复合物并且不稳定;我们假设这解释了其在人类中的无效表型。负染电镜下Seipin呈环状。我们推测，seipin至少在组织液滴或液滴和ER之间的通信中起着结构性作用。
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.
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