The isoprenoid pathway from mevalonate generates several products important for retinal photoreceptor cell function. The primary product, cholesterol, represents about 2% of the tissue weight and about 6-8% of the total rod outer segment (ROS) membrane lipid. A second product, dolichyl phosphate (Dol-P), is required for oligosaccharide transfer in N-linked glycoprotein synthesis, a process essential for the integration of opsin into the ROS membrane. Very little is known regarding the metabolism of these two lipids in the retina. The present proposal is aimed at alleviating this deficiency. The chemical levels of cholesterol, squalene, Dol-P and dolichol will be determined by high performance liquid chromatography. The role of de novo synthesis in the incorporation of isoprenoids into the retina and ROS will be determined by measuring the absolute rates of synthesis in isolated retina using 3H20 and (3H)acetate. The requirement for isoprenoid synthesis for the (a) assembly of ROS membranes, (b) incorporation of opsin into ROS, and (c) glycosylation of opsin will be examined using inhibitors which act at specific enzymatic steps of the de novo pathway of isoprenoid biosynthesis. Finally, to address the question of whether isoprenoids and opsin are routed by different pathways, the dependence of functioning Golgi-derived vesicular traffic on the transport of isoprenoids to the ROS will be determined. The above studies should yield considerable information concerning the role and requirement of de novo isoprenoid synthesis for the assembly and function of ROS membranes.
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