Molecular cloning, membrane topology, and localization of bovine rom-1 in rod and cone photoreceptor cells.

PURPOSE To characterize the molecular properties, cellular distribution, and subcellular distribution of bovine rom-1 and its interaction with peripherin/rds in photoreceptor cells as an important step toward elucidating the role of rom-1 in photoreceptor outer segment structure-function relationships and in inherited retinal degenerative disorders. METHODS Bovine rom-1 cDNA, including a portion of the promoter region, was cloned, sequenced, and heterologously expressed in CHO-K1 and COS-1 cultured mammalian cells. Monoclonal and polyclonal antibodies to specific regions of bovine rom-1 were generated and used with biochemical and immunocytochemical techniques to study the membrane topology, subcellular distribution, and interaction of rom-1 with peripherin/rds. RESULTS The primary structure of bovine rom-1 is highly homologous to that of human and mouse rom-1. Proteolytic digestion studies and immunolabeling studies of rom-1 in rod outer segment membranes indicate that the C-terminus of rom-1 is localized to the cytoplasmic side and that a large segment is localized to the lumen side of the disc membrane. Post-embedding and pre-embedding immunogold labeling studies for electron microscopy show that rom-1 is localized to the rim region of bovine rod outer segment disc membranes; rom-1 or a closely related homologue also is present at the rim region of cone outer segment disc membranes. Immunofluorescence studies of mammalian cells expressing rom-1 indicate that rom-1 is not translocated to the plasma membrane but instead is retained in internal cellular membranes. Immunoprecipitation studies indicate that all rom-1 and peripherin/rds from rod outer segments form a tightly associated complex. CONCLUSIONS Rom-1 and peripherin/rds are two structurally related protein subunits of an integral membrane protein complex found on the rim region of rod outer segment disc membranes. Rom-1 or a homologue also is present in the rim region of cone outer segment disc membranes, where it most likely associates with peripherin/rds to form a membrane protein complex.