title: Structural and functional analysis of the native peripherin-ROM1 complex isolated from photoreceptor cells.
abstract: Peripherin and its homologue ROM1 are retina-specific members of the tetraspanin family of integral membrane proteins required for morphogenesis and maintenance of photoreceptor outer segments, regions that collect light stimuli. Over 100 pathogenic mutations in peripherin cause inherited rod- and cone-related dystrophies in humans. Peripherin and ROM1 interact in vivo and are predicted to form a core heterotetrameric complex capable of creating higher order oligomers. However, structural analysis of tetraspanin proteins has been hampered by their resistance to crystallization. Here we present a simplified methodology for high yield purification of peripherin-ROM1 from bovine retinas that permitted its biochemical and biophysical characterization. Using size exclusion chromatography and blue native gel electrophoresis, we confirmed that the core native peripherin-ROM1 complex exists as a tetramer. Peripherin, but not ROM1, is glycosylated and we examined the glycosylation site and glycan composition of ROM1 by liquid chromatographic tandem mass spectrometry. Mass spectrometry was used to analyze the native complex in detergent micelles, demonstrating its tetrameric state. Our electron microscopy-generated structure solved to 18 Å displayed the tetramer as an elongated structure with an apparent 2-fold symmetry. Finally, we demonstrated that peripherin-ROM1 tetramers induce membrane curvature when reconstituted in lipid vesicles. These results provide critical insights into this key retinal component with a poorly defined function.
authors.affiliation: From the Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4965 and.
authors.forename: Brian M
authors.initials: BM
authors.lastname: Kevany
authors.forename: Yaroslav
authors.initials: Y
authors.lastname: Tsybovsky
authors.forename: Iain D G
authors.initials: ID
authors.lastname: Campuzano
authors.forename: Paul D
authors.initials: PD
authors.lastname: Schnier
authors.forename: Andreas
authors.initials: A
authors.lastname: Engel
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Palczewski
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D006031:Glycosylation; D008081:Liposomes / Q000737:chemistry; D008969:Molecular Sequence Data; D064531:Peripherins / Q000737:chemistry* / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry; D055503:Protein Multimerization; D055214:Retinal Photoreceptor Cell Outer Segment / Q000737:chemistry / Q000378:metabolism*; D059470:Tetraspanins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D008081:Liposomes; D064531:Peripherins; D011134:Polysaccharides; D059470:Tetraspanins
keywords: Retina; Retinal Degeneration; Retinal Metabolism; Vision; Vitamin A