Super-resolution microscopy reveals a Rab6a-dependent trafficking hub for rhodopsin at the mammalian rod photoreceptor Golgi

Rod photoreceptor stability is critical for retinal health and lifelong vision. Rhodopsin (Rho) trafficking is essential for rod homeostasis, as its mislocalization precedes rod cell death in inherited retinal disorders such as retinitis pigmentosa. Despite its importance, the molecular mechanisms of Rho trafficking in mammalian rods remain largely undefined. We investigated Rho's subcellular organization in the mammalian rod Golgi complex. We utilized STORM and structured illumination microscopy super-resolution imaging to map Golgi proteins with Rho in mouse and macaque rods. Our analysis found that a large proportion of Rho in this subcellular region colocalizes with Rab6a in the trans-Golgi. To functionally test this interaction, we utilized a dominant-negative Rab6a mutant in HEK293T cells and mouse rods. The mutant significantly inhibits Rho secretion in cell culture, causing intracellular retention. In mouse rods, the mutant similarly causes significant trans-Golgi Rho retention; however, a majority of Rho protein still escaped the Golgi and reached the outer segment. Together, these findings uncover critical new subcellular details about Rho organization at the Golgi and establish a role for Rab6a as a regulator of Rho protein release from the trans-Golgi in mammalian rods. Our results provide critical insight into the protein trafficking mechanisms essential for long-term photoreceptor health.