Project Summary Rhodopsins are G-protein coupled proteins that initiate the visual signal transduction cascade in response to light exposure. The mechanism of Rhodopsin homeostasis draws research interest in part because dysfunctional Rhodopsins are among the most frequent causes of Retinitis Pigmentosa (RP), a genetic disorder with age-related retinal degeneration. Among those associated with RP are Rhodopsin mutants with impaired protein folding properties. Rhodopsin undergoes synthesis and folding in the endoplasmic reticulum (ER), and excessive misfolding of Rhodopsin could impose stress on this organelle. In response to ER stress, cells activate an adaptive signaling response that regulates gene expression, widely referred to as the Unfolded Protein Response (UPR). Among others, UPR signaling induces the expression of genes that help to fold or degrade misfolded proteins in the ER. One of the UPR signaling branches is mediated by the ER stress sensor PERK and its downstream effector ATF4. The basic mechanisms of UPR signaling, Rh1 homeostasis, and retinal degeneration are conserved in Drosophila melanogaster. Specifically, Drosophila ninaE encodes the Rhodopsin-1 (Rh1) protein expressed in adult eye photoreceptors. A mutant allele of this gene, ninaEG69D, serves as a model for RP as it imposes ER stress, activates the UPR, and dominantly causes age-related retinal degeneration. The long-term goal of this project is to harness the genetic and genomic tools of Drosophila to understand the role of UPR in retinal degeneration. Our preliminary studies based on this approach suggest a need to significantly revise our conventional view of the UPR in retinal degeneration. In Specific Aims 1 and 2, I propose to re-evaluate the idea that ATF4 is the primary downstream effector of PERK-mediated UPR. Arguing against this conventional notion, we recently found that a bZIP transcription factor Xrp1 mediates ATF4-independent PERK signaling. We will determine how Xrp1 is regulated during UPR, and how they affect retinal degeneration in the Drosophila RP model. In addition, we plan to test the idea that the C/EBP family transcription factor IRBP18 works together with Xrp1 to mediate the UPR in the Drosophila RP model. We further propose to identify the human equivalent of the Xrp1/IRBP18 complex. In Aim 3, I propose to investigate how Rh1 is degraded in the Drosophila models of RP. Until now, there has been much focus on the ubiquitin ligases at the ER that can help degrade misfolded proteins. Unexpectedly, our photoreceptor-specific gene expression profiling studies reveal that many endosomal trafficking regulators are induced in ninaEG69D/+ photoreceptors. Based on this, we plan to test whether these endosomal factors mediate the degradation of Rh1 in these photoreceptors. We further propose to test if this newly found ER stress-endosome link affects the course of retinal degeneration in this RP model. A successful outcome of these plans may help significa...