Heterologous expression of photoreceptor peripherin/rds and Rom-1 in COS-1 cells: assembly, interactions, and localization of multisubunit complexes.
Peripherin/rds is a 39 kDa integral membrane glycoprotein essential for normal photoreceptor cell development in vertebrates. It has been implicated in several human retinal degenerative diseases including retinitis pigmentosa and macular degeneration and is thought to play a structural role at photoreceptor outer segment disk rims, where it forms a tightly-associated complex with rom-1, a nonglycosylated 37 kDa homologue. Western blot analysis of COS-1 cells transiently transfected with full-length cDNA coding for either peripherin/rds or rom-1 indicates that each protein is expressed primarily as a disulfide-linked homodimer; recombinant peripherin/rds is glycosylated while recombinant rom-1 is not--akin to their counterparts in rod photoreceptor disk membranes. Upon cotransfection of the two cDNAs, the specific assembly of a stable peripherin/rds--rom-1 complex is observed. Immunofluorescence microscopy studies demonstrate that both singly and coexpressed peripherin/rds and rom-1 complexes are localized primarily within internal membranes of transfected cells. Velocity sedimentation data indicate that the recombinant complexes (4.9 S) are assembled with a subunit stoichiometry similar to those extracted from ROS membranes (4.5 S) and are most consistent with a tetrameric arrangement of polypeptides. Sedimentation analyses of individually expressed peripherin/rds (5.1 S) and rom-1 (4.3 S) suggest that each polypeptide can also assemble into a tetrameric form in the absence of its homologue partner. Subunit assembly and interactions are discussed in terms of their potential role in hereditary retinal diseases.
Goldberg
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AF
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, Canada.
Moritz
O L
OL
Molday
R S
RS
D000818:Animals; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D005136:Eye Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007381:Intermediate Filament Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009419:Nerve Tissue Proteins; D064531:Peripherins; D010786:Photoreceptor Cells / Q000378:metabolism*; D011233:Precipitin Tests; D059470:Tetraspanins
D005136:Eye Proteins; D007381:Intermediate Filament Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; C578994:PRPH protein, human; C578997:PRPH2 protein, human; D064531:Peripherins; C513106:ROM1 protein, human; D059470:Tetraspanins