A Tandem Fluorescent Rh1::eGFP mCherry Reporter Reveals Rhodopsin 1 Trafficking and Turnover In Vivo in Drosophila Photoreceptors
Rhodopsin 1 (Rh1), the major visual pigment of Drosophila photoreceptors, requires a vitamin A-derived chromophore for proper folding, maturation and transport to the rhabdomere. While the endocytic trafficking of mature, light-activated Rh1 to endolysosomal compartments is well characterized, the fate of immature Rh1 during defective ER maturation remains unclear. Here, we developed a Rh1::eGFP-mCherry tandem reporter to monitor Rh1 localization and compartment acidification in vivo. Exploiting the acid stability of mCherry, this reporter enables visualization of Rh1 after delivery to acidic environments and complements conventional antibody-based approaches. Under physiological conditions, pH-dependent quenching of eGFP fluorescence distinguished rhabdomeric Rh1 from Rh1 in acidic endolysosomal compartments. In vitamin A-deficient flies, immature apo-Rh1 accumulated within Calnexin-positive ER membranes, where we identified discrete acidified subdomains characterized by loss of eGFP fluorescence while mCherry fluorescence persisted. These structures remained continuous with the ER but were distinct from conventional endolysosomal compartments, revealing previously unrecognized acidified ER domains associated with immature Rh1. Together, our findings identify acidified ER subdomains as a previously unrecognized stage in Rh1 quality control and establish Rh1::eGFP-mCherry as a robust in vivo reporter for investigating rhodopsin trafficking, maturation and turnover under physiological and disease-relevant conditions.