Project Summary Rhodopsins are G-protein coupled proteins that initiate signal transduction in response to light exposure. There is significant interest in understanding Rhodopsin homeostasis 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. Because Rhodopsins undergo synthesis and folding in the endoplasmic reticulum (ER), such Rhodopsin mutants could impose stress on this organelle. Those conditions activate an adaptive signaling response that regulates gene expression, widely referred to as the Unfolded Protein Response (UPR). One particular UPR signaling branch relevant to this proposal is the one mediated by the ER stress sensor PERK and its downstream effector ATF4. Among others, UPR signaling induces the expression of genes that help fold or degrade misfolded proteins in the ER, thereby affecting retinal degeneration in RP. 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. Here, I propose to investigate new UPR signaling branches that may significantly change our understanding of Rhodopsin homeostasis and retinal degeneration. In Specific Aims 1 and 2, I propose to re-evaluate the widespread idea that ATF4 is the primary downstream effector of PERK-mediated UPR. Arguing against this, we recently identified a new sub-branch of the PERK pathway mediated by Xrp1, a bZIP transcription factor. How Xrp1 is regulated and whether it affects retinal degeneration remains unclear. We will specifically test the hypothesis that Xrp1 is translationally induced by PERK. We will determine if such induction affects the course of retinal degeneration and whether Xrp1 requires heterodimerization partners to regulate some or all downstream target genes. I further propose to identify the human equivalent of the Xrp1 heterodimer complex. In Aim 3, I propose to investigate a possible link between ER stress and endosome trafficking in the RP model. Most UPR studies have focused on its role in ER homeostasis. However, our recent gene expression profiling results reveal that ninaEG69D/+ photoreceptors also induce many endosomal trafficking regulators. I propose to determine if those endosomal factors are induced by the UPR or by other unconventional signaling pathways. We will further determine if those pathways affect Rhodopsin homeostasis and the course of retinal degeneration in ...