The structural and functional properties of bovine rhodopsin from rod outer segment disk and plasma membranes were compared by high performance liquid chromatography (HPLC), mass spectrometric analyses, and in vitro rhodopsin phosphorylation assays. Disk and plasma membranes separated by a ricin gold-dextran affinity perturbation method were treated with trypsin or cynogen bromide, and the N-terminal and C-terminal rhodopsin peptides were isolated by immunoaffinity chromatography using antirhodopsin monoclonal antibodies coupled to Sepharose. Reverse phase HPLC chromatograms of the C-terminal and N-terminal peptides from disk and plasma membrane rhodopsin were found to be similar. Mass spectrometric, PicoTag, and hexose analyses of the tryptic 1-16 N-terminal peptides further indicated that the post-translational glycosylation of plasma membrane rhodopsin is identical to that of disk membrane rhodopsin. HPLC analysis of soluble peptides obtained from cyanogen bromide and tryptic digestion of immunoaffinity purified rhodopsin also indicated that no significant differences exist between disk and plasma membrane rhodopsin. Light-induced phosphorylation of rhodopsin in disk and plasma membranes were also compared using in vitro phosphorylation assays. Plasma membrane rhodopsin was found to undergo light-dependent, rhodopsin kinase catalyzed phosphorylation to the same extent as disk membrane rhodopsin. These results indicate that the bulk rhodopsin in rod outer segment plasma membranes appears to be identical to rhodopsin in disk membranes in regard to primary structure, post-translational glycosylation and light-dependent phosphorylation. On this basis, it is unlikely that the sorting of rhodopsin between disk and plasma membranes occurs by a mechanism based on differences in structural properties of rhodopsin.
Bibliographical noteFunding Information:
Correspondence to: R.S. Molday, Department of Biochemistry, Faculty of Medicine, 2146 Health Sciences Mall, Vancouver, B.C., V6T 1Z3, Canada. 1 Supported by grants from National Eye Institute (EY0 2422) and Medical Research Council of Canada (MT 5822). Y.T.H. is the recipient of a MRC studentship. 2 Present address: Department of Molecular Cellular and Develop-mental Biology, University of Colorado, Boulder, Colorado Disk and plasma membranes separated by affinity density perturbation methods have been shown to have different protein and lipid compositions [2-4]. The plasma membrane contains the 63 kDa cGMP-gated cation channel and an associated 240 kDa spectrin-like protein [5,6], the Na+/Ca2÷-K ÷ exchanger , membrane-associated glyceraldehyde-3-phosphate dehydrogenase , GLUT-1 glucose transporter , and other unidentified proteins . Disk membranes contain a high molecular weight rim protein [3,10], the periph-erin/rds protein , ROM-1 protein  and several other proteins . The lipid composition of the plasma membrane has been reported to have a high content of cholesterol, unsaturated Ct8 (18:2; 18:3) fatty acids, and the saturated C14 fatty acid, whereas the disk membranes are high in the saturated C 18 fatty acid and unsaturated C22 (22:6) docosahexaenoic acid .
- Disk membrane
- Monoclonal antibody
- Plasma membrane
- Rhodopsin phosphorylation