Crag is a GEF for Rab11 and regulates Rhodopsin trafficking in adult photoreceptor cells. Bo Xiong^1*, Manish Jaiswal², Ke Zhang³, Hector Sandoval⁴, Wu-Lin Charng¹, Tongchao Li¹, Gabriela David¹, Shinya Yamamoto^1,4, Hugo Bellen^1,2,3,4,5,6. 1) PROGRAM IN DVELOPMENTAL BIOLOGY, BAYLOR COLLEGE OF MEDICINE, HOUSTON, TX; 2) Howard Hughes Medical Institute, Baylor College of Medicine, Houston, TX; 3) Program in Structural and Computational Biology and Molecular Biophysics, Baylor College of Medicine, Houston, TX; 4) Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX; 5) Department of Neuroscience, Baylor College of Medicine, Houston, TX; 6) Neurological Research Institute, Baylor College of Medicine, Houston, TX.

   Rhodopsins (Rh) are G-protein coupled light sensors, and Rh1 is the major Rh in Drosophila which is mainly localized to the rhabdomere membrane. Upon photoactivation, a fraction of Rh1 is internalized and degraded, but it remains unclear how the rhabdomeric Rh1 pool is replenished and what molecular players are involved. In this study, we show that Crag, a DENN domain containing protein, is required for the homeostasis of Rh1 upon light exposure. The absence of Crag causes a light induced accumulation of cytoplasmic Rh1 and vesicles, leading to a retinal degeneration in adult flies. When endocytosis of Rh1 is triggered by blue light, Rh1 internalization and degradation is not affected in Crag mutant cells. However, a persistent accumulation of Rh1 is observed in Crag mutant cells but not in wild type cells after a recovery period. The accumulated Rh1 is partially associated with the trans-Golgi compartment. We therefore conclude that Crag is required for post-Golgi trafficking of newly synthesized Rh1. Furthermore, we show that Crag is a guanine nucleotide exchange factor for Rab11. Knockdown of Rab11 leads to a similar light dependent retinal degeneration phenotype and overexpression of a constitutive active form of Rab11 partially rescues the defects associated with loss of Crag. We propose that upon light stimulation, Crag is required for trafficking of Rhodopsin from the Trans-Golgi network to rhabdomere membranes via a Rab11 dependent vesicular transport.