An in cifro system for the biochemical and autoradiographic study of rhodopsin biosynthesis has been developed. Using the isolated frog retina, the incorporation of radioactive leucine into rod outer segment rhodopsin was measured under a variety of experimental conditions. Incorporation of leucine into rhodopsin was shown to increase linearly with time and with increasing isotope concentration, and was sensitive to puromycin and oligomycin, but not chloramphenicol. When measured at various temperatures from 0 to 34", maximal incorporation occurred at 26". Reduced incorporation occurred when the retinas were incubated in phosphate-Ringer medium containing 15 mM phosphate when compared to bicarbonate-Ringer containing 1.25 mM phosphate. Incorporation was also reduced by concentra-T h e vertebrate retina contains SDecialized cells which are responsible for receiving light energy and translating it into neurochemical impulses. These cells, the photoreceptors, are composed of two nearly separate segments, each performing different functions in the cell. The inner segment is responsible for biosynthesis, metabolism, and maintenance of the cell, and contains the cell nucleus, the Golgi apparatus, endoplasmic reticulum, and a dense group of mitochondria which are located at its distal end. The cylindrical outer segment contains the light-trapping organelles, a stack ,of hundreds of lamellar membrane disks containing the visual pigment. These disks are separate in the rod outer segment, but continuous in the cone.The rod outer segment (ROS)l undergoes continuous renewal by assembling new disks at the base of the outer segment, a process which displaces the old disks toward the pigment epithelium (Young, 1967;Young and Droz, 1968;Young and Bok, 1969). To maintain the rod outer segment at a uniform length, the outermost disks are continuously shed into the pigment epithelium (Young and Bok, 1969). Autoradiographic and biochemical experiments using labeled amino acids injected in ciao have shown that protein for new disks is synthesized in the inner segment and subsequently transported to the outer segment where disk assembly occurs (Young and Bok, 1969;Hall et al., 1969).Since each frog photoreceptor cell makes about 36 new $ Supported by a postdoctoral fellowship from the National Institutes of H ealth. Abbreviations used are: RBG, Krebs-Ringer bicarbonate incubation medium; wash buffer, 0.067 M sodium phosphate-4 mM MgCI: (pH 7.0); ROS, rod outer segment; RR, retinal residues; CVP, crude visual pigment; O S , outer segment insoluble; RRS, retinal residue soluble; RRI, retinal residue insoluble; ft3/hr, standard cubic feet per hour. 1996 B I O C H E M I S T R Y , V O L . 1 2 , N O . 1 0 , 1 9 7 3tions of glucose greater than 20 mM and if casamino acids were added or if glycerol replaced glucose. Incubation was normally carried out in the dark using dark adapted, pigment epithelium-free retinas. If the retinas were partially bleached prior to incubation and incubated in the dark, no differences in incorporation of leucine ...