HETEROLOGOUS EXPRESSION OF PHOTORECEPTOR PERIPHERIN RDS AND ROM-1 IN COS-1 CELLS - ASSEMBLY, INTERACTIONS, AND LOCALIZATION OF MULTISUBUNITCOMPLEXES/

Peripherin/rds is a 39 kDa integral membrane glycoprotein essential fo r normal photoreceptor cell development in vertebrates. It has been im plicated in several human retinal degenerative diseases including reti nitis pigmentosa and macular degeneration and is thought to play a str uctural role at photoreceptor outer segment disk rims, where it forms a tightly-associated complex with rom-1, a nonglycosylated 37 kDa homo logue. Western blot analysis of COS-1 cells transiently transfected wi th full-length cDNA coding for either peripherin/rds or rom-1 indicate s that each protein is expressed primarily as a disulfide-linked homod imer; recombinant peripherin/rds is glycosylated while recombinant rom -1 is not-akin to their counterparts in rod photoreceptor disk membran es. Upon cotransfection of the two cDNAs, the specific assembly of a s table peripherin/rds(-)rom-1 complex is observed. Immunofluorescence m icroscopy studies demonstrate that both singly and coexpressed periphe rin/rds and rom-l complexes are localized primarily within internal me mbranes of transfected cells. Velocity sedimentation data indicate tha t the recombinant complexes (4.9 S) are assembled with a subunit stoic hiometry similar to those extracted from ROS membranes (4.5 S) and are most consistent with a tetrameric arrangement of polypeptides. Sedime ntation analyses of individually expressed peripherin/rds (5.1 S) and rom-1 (4.3 S) suggest that each polypeptide can also assemble into a t etrameric form in the absence of its homologue partner. Subunit assemb ly and interactions are discussed in terms of their potential role in hereditary retinal diseases.