MOLECULAR-CLONING, MEMBRANE TOPOLOGY, AND LOCALIZATION OF BOVINE ROM-1 IN ROD AND CONE PHOTORECEPTOR CELLS

Purpose. To characterize the molecular properties, cellular distributi on, and subcellular distribution of bovine rom-l and its interaction w ith 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 disorder s. Methods. Bovine rom-l cDNA, including a portion of the promoter reg ion, was cloned, sequenced, and heterologously expressed in CHO-KI and COS-1 cultured mammalian cells, Monoclonal and polyclonal antibodies to specific regions of bovine rom-l were generated and used with bioch emical and immunocytochemical techniques to study the membrane topolog y, subcellular distribution, and interaction of rom-l with peripherin/ rds. Results. The primary structure of bovine rom-l is highly homologo us to that of human and mouse rom-l. Proteolytic digestion studies and immunolabeling studies of rom-l in rod outer segment membranes indica te that the G-terminus of rom-l is localized to the cytoplasmic side a nd that a large segment is localized to the lumen side of the disc mem brane. Post-embedding and pre-embedding immunogold labeling studies fo r electron microscopy show that rom-l is localized to the rim region o f bovine rod outer segment disc membranes; rom-l or a closely related homologue also is present at the rim region of cone outer segment disc membranes. Immunofluorescence studies of mammalian cells expressing r om-l indicate that rom-l is not translocated to the plasma membrane bu t instead is retained in internal cellular membranes. Immunoprecipitat ion studies indicate that all rom-l and peripherin/rds from rod outer segments form a tightly associated complex. Conclusions. Rom-l and per ipherin/rds are two structurally related protein subunits of an integr al membrane protein complex found on the rim region of rod outer segme nt disc membranes. Rom-l or a homologue also is present in the rim reg ion of cone outer segment disc membranes, where it most likely also as sociates with peripherin/rds to form a membrane protein complex.