Seipin is a discrete homooligomer.
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 H(2)O, and D(2)O 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.
Department of Pharmacology, The University of Texas Southwestern Medical Center, Dallas,Texas 75390, United States.
Derk
D
Binns
Sungkyung
S
Lee
Christopher L
CL
Hilton
Qiu-Xing
QX
Jiang
Joel M
JM
Goodman
D002850:Chromatography, Gel; D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism*; D044388:GTP-Binding Protein gamma Subunits / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D055503:Protein Multimerization; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
C432679:BSCL2 protein, human; D005656:Fungal Proteins; D044388:GTP-Binding Protein gamma Subunits; D029701:Saccharomyces cerevisiae Proteins; C554737:seipin protein, S cerevisiae