Identification of additional outer segment targeting signals in zebrafish rod opsin

In vertebrate photoreceptors, opsins are highly concentrated in a morphologically distinct ciliary compartment known as the outer segment (OS). Opsin is synthesized in the cell body and transported to the OS at a remarkable rate of 100-1000 molecules per second. Opsin transport defects contribute to photoreceptor loss and blindness in human ciliopathies. Previous studies revealed that the opsin C-terminal tail, of 44 amino acids, is sufficient to mediate OS targeting in Xenopus photoreceptors. Here we show that although the Xenopus C-terminus retains this function in zebrafish, the homologous zebrafish sequence is not sufficient to target opsin to the OS. This functional difference is largely caused by a change of a single amino acid present in Xenopus, but not in other vertebrates examined. Furthermore, we find that sequences in the 3rd intracellular cytoplasmic loop (IC3) and adjacent regions of transmembrane helixes 6 and 7 are also necessary for opsin transport in zebrafish. Combined with the cytoplasmic tail, these sequences are sufficient to target opsin to the ciliary compartment.