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Molecular cloning, localization, and heterologous expression of bovine rom-1, a photoreceptor disc rim specific protein

University of British Columbia

Rom-1 is an integral membrane protein found in mammalian rod photoreceptor disc membranes. Much interest in rom-1 has been generated by its structural similarity to peripherin/rds, a protein implicated in a number of inherited retinal degenerative disorders. Defects in the ROM1 gene have also been associated with a digenic form of retinal degeneration, in which changes in the sequence of both rom-1 and peripherin/rds are necessary to produce the retinal degeneration phenotype. As an essential step in studying structure-function relationships of rom-1 in photoreceptor membranes, the cDNA encoding the bovine homolog of rom-1 was cloned and sequenced. The coding sequence was obtained from a cDNA library, while upstream and promoter sequences were obtained by inverse polymerase chain reaction. The sequence is 84% identical to human rom-1 and 32% identical to bovine peripherin/rds at the amino acid level. The predicted membrane topology of bovine rom- 1 and a number of short motifs found within the sequence indicate that bovine rom-1 is a member of the transmembrane 4 super family (TM4SF) group of proteins. Short segments of bovine rom-1 were expressed in E. coli as fusion proteins, which were then used as immunogens to generate monoclonal and polyclonal antibodies to specific regions of bovine rom-1. The antibodies were used as probes to study the membrane topology, subcellular distribution and interaction of rom-1 with peripherin/rds. The results indicate that rom-1 and peripherin/rds are subunits of a membrane protein complex that is localized to the rim regions of photoreceptor outer segment disc membranes. In contrast to previous reports, rom-1 was detected in cone photoreceptors as well as rod photoreceptors. These results are discussed in terms of the role of rom-1 in disc structure and retinal degenerative diseases. Bovine rom-1 was heterologously expressed in COS-1 cells. The heterologously expressed protein was characterized using immunocytochemistry techniques. Heterologously expressed rom-1 is similar to rom-1 from ROS membranes in terms of glycosylation and subunit interactions with itself and with peripherin/rds. A number of mutant forms of rom-1 were also examined using the heterologous expression system in order to determine whether intracellular distribution or subunit interactions are altered relative to wild-type rom-1. Truncation of rom-1 after the fourth transmembrane domain was found to increase expression levels and did not affect interaction with peripherin/rds. The mutation romL188P was found to alter the quaternary structure of rom-1, as determined by velocity sedimentation. These studies provide valuable new information concerning the interaction of rom-1 and peripherin/rds subunits in photoreceptor outer segment membranes. Additionally, a system is described for studying the interaction of wild-type and mutant forms of these subunits heterologously expressed in COS-1 cells. Finally, the discovery that rom-1 is present in cone photoreceptors as well as rod photoreceptors proves that the cone-dominant and rod-dominant phenotypes associated with different RDS mutations are not due to absence of the rom-1 subunit in cone photoreceptors.

Thesis/Dissertation

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2009-03-17

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http://hdl.handle.net/2429/6128

Biochemistry and Molecular Biology

University of British Columbia

UBCV

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