Tunicamycin blocks the incorporation of opsin into retinal rod outer segment membranes.
Isolated frog retinas were incubated with radiolabeled glycoprotein precursors in the presence or absence of tunicamycin (TM), a selective inhibitor of protein N-glycosylation. In dual-label incubations, TM inhibited the incorporation of [3H]mannose into total retina Cl3CCOOH-precipitable material by 85% relative to controls, whereas incorporation of [14C]leucine was not significantly affected. In a companion single-label incubation, TM blocked the incorporation of [3H]leucine into rod outer segment (ROS) membrane Cl3CCOOH-precipitable material by 95% relative to controls. When retinas were labeled with [35S]methionine, fluorograms of NaDodSO4/polyacrylamide gels from control retinas and ROS membranes exhibited a heavily labeled component (apparent Mr approximately 37,000) which had the electrophoretic and antigenic properties of opsin, the rod visual pigment apoglycoprotein. TM-treated retinas exhibited a substantially reduced labeling of the Mr 37,000 component and incorporation of label into a component (apparent Mr approximately 32,000) not found in control retinas, which exhibited the electrophoretic and antigenic behavior of nonglycosylated opsin. ROS membranes isolated from TM-treated retinas contained neither the Mr 37,000 nor the Mr 32,000 radiolabeled species. Light-microscope autoradiograms of retinas incubated with [3H]leucine in the absence of TM exhibited bands of silver grains at the base of ROS, indicative of new membrane assembly. However, no such bands were observed in autoradiograms of TM-treated retinas. These results suggest that glycosylation of opsin is required for its incorporation into ROS membranes.
Fliesler
S J
SJ
Basinger
S F
SF
D000818:Animals; D005136:Eye Proteins / Q000302:isolation & purification / Q000378:metabolism*; D005944:Glucosamine / Q000031:analogs & derivatives*; D006027:Glycosides / Q000378:metabolism; D066298:In Vitro Techniques; D008566:Membranes / Q000378:metabolism; D008970:Molecular Weight; D010786:Photoreceptor Cells / Q000378:metabolism*; D011894:Rana pipiens; D012374:Rod Cell Outer Segment / Q000187:drug effects / Q000378:metabolism*; D017299:Rod Opsins; D014415:Tunicamycin / Q000494:pharmacology*
D005136:Eye Proteins; D006027:Glycosides; D017299:Rod Opsins; D014415:Tunicamycin; D005944:Glucosamine