Molecular cloning, membrane topology, and localization of bovine rom-1 in rod and cone photoreceptor cells.
OBJECTIVE
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.
Moritz
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Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, Canada.
Molday
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D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D015153:Blotting, Western; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure*; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005136:Eye Proteins / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure*; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation; D006801:Humans; D007381:Intermediate Filament Proteins / Q000378:metabolism / Q000648:ultrastructure; D008562:Membrane Glycoproteins / Q000378:metabolism / Q000648:ultrastructure; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure*; D051379:Mice; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins; D064531:Peripherins; D010786:Photoreceptor Cells / Q000378:metabolism / Q000648:ultrastructure*; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D059470:Tetraspanins
D000911:Antibodies, Monoclonal; 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; C578996:Prph2 protein, mouse; C513106:ROM1 protein, human; C081477:Rom1 protein, mouse; D059470:Tetraspanins; D004247:DNA