Membrane biosynthesis in the frog retina: opsin transport in the photoreceptor cell.
Rhodopsin biosynthesis and transport in the photoreceptor cell have been analyzed by subcellular fractionation of frog retinas after short periods of radioactive amino acid incorporation in vivo. Labelled membrane proteins were identified by autoradiography or sodium dodecyl sulfate-polyacrylamide gels. One of the most intensly labeled proteins in retina had a molecular weight comparable to opsin isolated from purified rod outer segments (ROS). Incorporation of label into this protein was rapid; the relative specific activity then diminished after the first 2 hr as radioactivity was transferred from retinal subcellular fractions to ROS. The kinetics of this transfer resembled rates previously observed by Hall et al. (Hall, M. O., Bok, D., and Bacharach, A.C.E. (1969), J. Mol. Biol. 45, 397). To identify the rapidly labeled protein as opsin we devised a new technique of two-dimensional immunoelectrophoresis of detergent solubilized membrane proteins. Antibodies were prepared against both whole ROS and opsin. After initial separation of retinal proteins on sodium dodecyl sulfate-polyacrylamide gels, a second dimension of electrophoresis in agarose, containing antisera, resulted in the formation of specific immunoprecipitates. Immunochemical analysis of all membranous and soluble retinal subcellular fractions indicated that newly synthesized opsin was membrane bound upon completion of synthesis. At no period of incorporation was a soluble form of newly synthesized opsin detectable. On this basis, we suggest that this protein is apparently transported as a water-insoluble membrane-bound molecule through the cytoplasm or along membranes of the inner segment to its assembly site near the base of the outer segment.
D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D007122:Immunoelectrophoresis; D007700:Kinetics; D008854:Microscopy, Electron; D010786:Photoreceptor Cells / Q000378:metabolism*; D011817:Rabbits / Q000276:immunology; D011894:Rana pipiens; D011898:Ranidae; D012160:Retina / Q000378:metabolism* / Q000648:ultrastructure; D012168:Retinal Pigments / Q000378:metabolism*; D012243:Rhodopsin / Q000378:metabolism
D000596:Amino Acids; D012168:Retinal Pigments; D012243:Rhodopsin