Project Summary The long-term goal of the proposed research is to improve the understanding of the molecular mechanisms by which mutations in genes that regulate rhodopsin trafficking and photoreceptor membrane renewal lead to retinal diseases. Mutations in rhodopsin that affect its targeting motif cause severe forms of autosomal dominant retinitis pigmentosa (ADRP). In the course of our research, we defined the rhodopsin C-terminal VxPx motif as a conserved ciliary targeting signal. Furthermore, we identified a ciliary targeting complex that recognizes this signal and regulates sorting into post-Golgi rhodopsin transport carriers (RTCs). Our previous studies revealed that membrane targeting to the rod outer segments (ROS) is a conserved form of ciliary targeting, and that the complex we identified is a conserved complex that targets sensory receptors to primary cilia through intricate functional networks of small GTPases and their regulators that are exquisitely sensitive to mutations causing retinal degenerations and ciliopathies. We now propose to define the function of the Rab8 GEF Rabin8, and determine whether and how its phosphorylation by NDR2/STK38L kinase, a product of (erd) retinal degeneration gene, controls rhodopsin trafficking and ciliogenesis. We will also delineate the molecular mechanisms regulating Rabin8 interactions with the R-SNARE VAMP7, and determine how their dysfunction affects ROS membrane trafficking. To accomplish this, we will use our established cell-free system that reconstitutes rhodopsin trafficking in vitro and probe molecular interactions by kinase assays, and pulldown experiments with recombinant and purified components—as well as by transgenic expression of mutant and phosphomimetic protein constructs, confocal microscopy, Correlative Light and Electron Microscopy (CLEM), Super Resolution (SR) direct Stochastic Optical Reconstruction Microscopy (dSTORM) imaging, and immunoprecipitation from transgenic retinas with anti-GFP antibody followed by LC-MS/MS analysis. The basic understanding of the molecular linkage between the regulatory machineries involved in the renewal of light-sensitive membranes through ciliary targeting and a wide range of systemic cilia disorders is expected to provide the foundation for improved therapeutic strategies to treat retina-specific and syndromic forms of photoreceptor loss.