The acylation of rat rhodopsin in vitro and in vivo

Rat retinas were incubated with [3H] palmitate and [14C] leucine and subsequently detergent-extracted. Glycoproteins were isolated on Con A-Sepharose columns and separated by gel electrophoresis. Leucine labeled the newly synthesized opsin but palmitate was esterified to both mature rhodopsin and newly synthesized opsin which migrated more slowly because of its untrimmed oligosaccharide chains. Crude rod outer segments were found to contain most of the palmitate-labeled mature rhodopsin, while the retinal debris contained most of the doubly labeled newly synthesized opsin. Homogenization of double-labeled retinas followed by centrifugation in a linear sucrose gradient gave rise to several bands of particles including purified rod outer segments, a Golgi-enriched fraction and a pellet enriched in endoplasmic reticulum. Newly synthesized opsin was first found in the pellet at the earliest incubation times and subsequently appeared in the Golgi fraction and finally in the rod outer segments. Palmitate-labeled mature rhodopsin was found only in the rod outer segments. It appeared at the earliest time points and increased with time. Thus the acylation of opsin occurs in the endoplasmic reticulum, shortly after polypeptide synthesis, and in the rod outer segments, the latter possibly as an exchange reaction. Most of the newly synthesized opsin remained in the pellet and did not pass through the Golgi to the rod outer segments. Intravitreal injection of [3H] palmitate and [14C] leucine gave rise to doubly labeled opsin that appeared to remain untrimmed for at least 6 hr in vivo. After 17 hr, both labels were found only in mature rhodopsin, thus accumulation of new molecules in the endoplasmic reticulum may occur in vivo. In addition, leucine maximally labeled the opsin-rhodopsin pool early in the first day whereas palmitate did not maximally label rhodopsin until 2- or 3 days post injection. Moreover, while leucine label was lost at day 9 because of rod outer-segment renewal and shedding, the palmitate label in rhodopsin remained unchanged. Thus, palmitate labeling in vivo reflects the pattern seen in vitro with a prolonged equilibration of rod outer-segment rhodopsin with the fatty-acid pool, probably mediated by a fatty acyl exchange reaction.