Structural and functional properties of rhodopsin from rod outer segment disk and plasma membrane.
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 cyanogen 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.
Hsu
Y T
YT
Department of Biochemistry, Faculty of Medicine, University of British Columbia, Vancouver, Canada.
Wong
S Y
SY
Connell
G J
GJ
Molday
R S
RS
D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D013058:Mass Spectrometry; D009898:Optic Disk / Q000737:chemistry*; D010766:Phosphorylation; D012243:Rhodopsin / Q000737:chemistry* / Q000302:isolation & purification; D012374:Rod Cell Outer Segment / Q000737:chemistry*
D012243:Rhodopsin