Vision begins when light is captured by the outer segment organelle of photoreceptor cells in the retina. Outer segments are modified cilia filled with hundreds of flattened disc-shaped membranes. Disc membranes are separated from the surrounding plasma membrane and each membrane type has unique protein components. The mechanisms underlying this protein sorting remain entirely unknown. In this study, we investigated the outer segment delivery of the rod cyclic nucleotide-gated (CNG) channel, which is located in the outer segment plasma membrane where it mediates the electrical response to light. Using Xenopus and mouse models of both sexes, we now show that the targeted delivery of the CNG channel to the outer segment utilizes the conventional secretory pathway, including protein processing in both ER and Golgi, and requires pre-assembly of its constituent α1 and β1 subunits. We further demonstrate that the N-terminal GARP domain of CNGβ1 contains two distinct functional regions. The glutamic acid-rich reg...