Tunicamycin-induced dysgenesis of retinal rod outer segment membranes. II. Quantitative freeze-fracture analysis

Tunicamycin (TM), a selective inhibitor of dolichylphosphate-dependent oligosaccharide biosynthesis, effectively blocks glycosylation, but not synthesis, of opsin, the rod visual pigment apoglycoprotein. In parallel with this inhibition, vesicular membrane material accumulates in the compartment between rod inner and outer segments (the intersegmental space) in TM-treated retinas (Fliesler et al, J Cell Biol 100:574-587, 1985). Freeze-fracture analysis was applied to isolated Xenopus laevis retinas which were incubated in the presence or absence of TM in order to clarify the relationship of those intersegmental membranous structures to others in the rod outer segment (ROS) assembly pathway. The membranes in the intersegmental space display characteristics similar to those of the ROS, but distinct from those of the inner segment. They exhibit densely particulate convex protoplasmic face (PF)-leaflets and relatively particle-free concave exoplasmic face (EF)-leaflets, similar to comparable leaflets of the ROS plasmalemma and nascent (immature) disc membranes. Quantitative analysis further demonstrates that the size distribution and densities of intramembrane particles (IMPs) in PF-leaflets of the intersegmental membranes are indistinguishable from those of ROS membranes, suggesting that both membrane systems contain similar integral proteins (i.e., opsin). Finally, the topology of the intersegmental membranes is most closely related to that of the ROS plasmalemma or nascent disc membranes, suggesting that they arise as a result of aberrant disc morphogenesis, rather than by breakdown of mature discs. Overall, the data support the conclusion that the tubulo-vesicular membranes in the intersegmental space represent newly assembled, opsin-containing material which has been efficiently compartmentalized in preparation for disc morphogenesis, but is incapable of forming normal, topologically closed discs.