Isolated Xenopus laevis retinas were incubated with 3H-labeled mannose or leucine in the presence or absence of tunicamycin (TM), a selective inhibitor of dolichyl phosphate-dependent protein glycosylation. At a TM concentration of 20 micrograms/ml, the incorporation of 3Hmannose and 3Hleucine into retinal macromolecules was inhibited by approximately 66 and 12-16%, respectively, relative to controls. Cellular uptake of the radiolabeled substrates was not inhibited at this TM concentration. Polyacrylamide gel electrophoresis revealed that TM had little effect on the incorporation of 3Hleucine into the proteins of whole retinas and that labeling of proteins (especially opsin) in isolated rod outer segment (ROS) membranes was negligible. The incorporation of 3Hmannose into proteins of whole retinas and ROS membranes was nearly abolished in the presence of TM. Autoradiograms of control retinas incubated with either 3Hmannose or 3Hleucine exhibited a discrete concentration of silver grains over ROS basal disc membranes. In TM-treated retinas, the extracellular space between rod inner and outer segments was dilated and filled with numerous heterogeneously size vesicles, which were labeled with 3Hleucine but not with 3Hmannose. ROS disc membranes per se were not labeled in the TM-treated retinas. Quantitative light microscopic autoradiography of retinas pulse-labeled with 3Hleucine showed no differences in labeling of rod cellular compartments in the presence or absence of TM as a function of increasing chase time. These results demonstrate that TM can block retinal protein glycosylation and normal disc membrane assembly under conditions where synthesis and intracellular transport of rod cell proteins (e.g., opsin) are not inhibited.
The Journal of Cell Biology – Rockefeller University Press
Published: Feb 1, 1985
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