In vivo identification of Drosophila rhodopsin interaction partners by biotin proximity labeling

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TL;DR

This study used a Rh1-TurboID fusion protein in Drosophila photoreceptors to identify in vivo interaction partners of rhodopsin via biotin proximity labeling. The method successfully identified known partners like arrestin 2 and visual signaling proteins, as well as novel candidates involved in rhodopsin maturation and transport, such as Rab32, Vap33, and PIP82. These findings provide a resource for understanding rhodopsin biology and potential mechanisms underlying retinal degeneration.

Full summary

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**Background:** Rhodopsins are G protein-coupled receptors that initiate visual transduction. In Drosophila, the major rhodopsin Rh1 interacts with G proteins and arrestins for signaling, and with chaperones and transport proteins for maturation and trafficking. Failures in these interactions lead to photoreceptor degeneration, making Drosophila a model for human retinal diseases. The study aimed to identify in vivo interaction partners of Rh1 using biotin proximity labeling with a TurboID fusion protein.

**Methods:** A Rh1::TbID fusion protein (Rh1 with C-terminal TurboID-V5) was expressed in Drosophila R1-6 photoreceptor cells under the Rh1 promoter. Transgenic lines were generated in wild-type (Rh1::TbID^wt^) and ninaE^17^ (Rh1::TbID^ninaE^) backgrounds. Expression was confirmed by immunoblotting with anti-Rh1 and anti-V5 antibodies. Rh1::TbID expression was 16% of native Rh1. Functionality was assessed by electroretinography (ERG) and immunohistochemistry. Biotinylated proteins were detected by streptavidin blots. For identification, biotinylated proteins were enriched by streptavidin pull-down, separated by SDS-PAGE, and analyzed by LC-MS/MS. Two approaches were used: in-gel digestion of excised bands and on-bead digestion. Stringent criteria (≥5 peptides, ≥3-fold enrichment over wild-type) were applied. Mutants (ninaE^17^, arr2^3^, ninaA^2^) were used to validate hits. Selected candidate mutants (PIP82, Rab32, Vap33, Kazachoc) were tested for Rh1 content by quantitative immunoblot.

**Key Results:** Rh1::TbID was functional: ERGs showed light responses and prolonged depolarization afterpotential (PDA) in Rh1::TbID^ninaE^ flies, though less pronounced due to reduced rhodopsin. Immunohistochemistry showed Rh1::TbID localized to rhabdomeres and underwent light-induced internalization. Streptavidin blots revealed biotinylated proteins at ~65 kDa (self-biotinylated Rh1::TbID), ~32 kDa (native Rh1), and a light-dependent 48 kDa band identified as arrestin 2. Arrestin 2 biotinylation occurred within 2 minutes of blue light and was reversible with red light. In arr2^3^ mutants, the 48 kDa band was absent. In ninaE^17^ mutants, the 32 kDa band was largely missing. In ninaA^2^ mutants, Rh1::TbID and arrestin 2 biotinylation were reduced. LC-MS/MS identified ~150 potential interaction partners. Table 1 lists 38 selected proteins, including phototransduction components (TRP, TRPL, PLCβ, arrestins, Gαq, Gβ76C, INAD, ePKC), rhabdomere structural proteins (Prominin, Chaoptin, NinaC), and proteins involved in maturation/transport (Calnexin 99A, EMC1, Rab32, Vap33, PIP82, Stardust, Clathrin, AP2, Dynamin, Flotillins, Rbcn). Biotinylated peptides were identified in 8 proteins, most prominently TRP (7 biotinylated peptides). In ninaA^2^ mutants, additional proteins were detected (DnaJ homolog, E3 UFM1-protein ligase, Pkc53E, inner nuclear membrane protein Man1). Quantitative immunoblot of mutants showed reduced Rh1 content in Vap33 (36% of control) and Kazachoc (38% of control), but not in PIP82 or Rab32 mutants.

**Clinical Implications:** This study establishes a method for identifying in vivo interaction partners of rhodopsin in Drosophila, a model for human retinal degenerations like Retinitis Pigmentosa. The identification of novel candidates (Rab32, Vap33, PIP82) and potential degradation pathways (ER-associated autophagy via DnaJ homolog in ninaA mutants) may reveal new targets for therapeutic intervention. The finding that Vap33 and Kazachoc mutants reduce Rh1 content suggests these proteins are important for rhodopsin stability. The method's limitation in densely packed microvilli (biotinylation radius ~10 nm) may cause non-specific labeling of abundant rhabdomeral proteins, but the light-dependent biotinylation of arrestin 2 argues for specificity.