The GTP- and Phospholipid-Binding Protein TTD14 Regulates Trafficking of the TRPL Ion Channel in Drosophila Photoreceptor Cells

Abstract

Recycling of signaling proteins is a common phenomenon in diverse signaling pathways. In photoreceptors of Drosophila, light absorption by rhodopsin triggers a phospholipase Cβ-mediated opening of the ion channels transient receptor potential (TRP) and TRP-like (TRPL) and generates the visual response. The signaling proteins are located in a plasma membrane compartment called rhabdomere. The major rhodopsin (Rh1) and TRP are predominantly localized in the rhabdomere in light and darkness. In contrast, TRPL translocates between the rhabdomeral plasma membrane in the dark and a storage compartment in the cell body in the light, from where it can be recycled to the plasma membrane upon subsequent dark adaptation. Here, we identified the gene mutated in trpl translocation defective 14 (ttd14), which is required for both TRPL internalization from the rhabdomere in the light and recycling of TRPL back to the rhabdomere in the dark. TTD14 is highly conserved in invertebrates and binds GTP in vitro. The ttd14 mutation alters a conserved proline residue (P75L) in the GTP-binding domain and abolishes binding to GTP. This indicates that GTP binding is essential for TTD14 function. TTD14 is a cytosolic protein and binds to PtdIns(3)P, a lipid enriched in early endosome membranes, and to phosphatidic acid. In contrast to TRPL, rhabdomeral localization of the membrane proteins Rh1 and TRP is not affected in the ttd14 P75L mutant. The ttd14 P75L mutation results in Rh1-independent photoreceptor degeneration and larval lethality suggesting that other processes are also affected by the ttd14 P75L mutation. In conclusion, TTD14 is a novel regulator of TRPL trafficking, involved in internalization and subsequent sorting of TRPL into the recycling pathway that enables this ion channel to return to the plasma membrane.

Immunocytochemical Labeling of Rhabdomeric Proteins in Drosophila Photoreceptor Cells Is Compromised by a Light-dependent Technical Artifact

“…(E) Immunocytochemical analysis on ommatidial cryosections from 1-3 day old flies was performed at well described time points of TRPL translocation (dark, 2 hr orange light, 16 hr orange light). 1,17,30 Cross sections through ommatidia are shown, except for row three which shows longitudinal sections for better visualization of TRPL vesicles. Cytoskeleton of rhabdomeres was visualized by Alexa Fluor 546 conjugated phalloidin (magenta).…”

“…Electroretinogram measurements were performed as described previously. 30 In brief, 12-day old flies were immobilized in improvised yokes made from pipette tips, before they were mounted in the center of a Faraday cage. Chlorinated silver wires were inserted into glass micropipettes filled with Davenport solution (100 mM NaCl 2 , 2 mM KCl, 1 mM CaCl 2 , 1.8 mM NaHCO 3 , pH 7.2) and utilized as electrodes.…”

Ectopic Expression of Mouse Melanopsin in Drosophila Photoreceptors Reveals Fast Response Kinetics and Persistent Dark Excitation

“…Immunocytochemistry of fly eyes was carried out as described previously (66) except that sections were additionally incubated after fixation on a shaker in 0.5% Triton X-100 in PBS (175 mM NaCl, 8 mM …”

Application of Fluorescent Proteins for Functional Dissection of the Drosophila Visual System

“…By way of labeling the repeating structure of ommatidia and the PRCs within them, FPs are highly useful to evaluate a possible impact of mutations on cell morphology, especially with respect to defects in the structure or spatial orientation of rhabdomeres. As reporters in the Drosophila eye, fusion proteins of GFP with different rhabdomerally located proteins such as rhodopsins, the ion channels TRP and TRPL, the signal regulator Arrestin 2 (Arr2) or the tail domain of the NINAC isoform p174 have been generated and expressed in PRCs (Figure 4) [25][26][27][28][29]. As sensory receptor cells, PRCs are highly specialized and precise regulation of their homeostasis is required in order to ensure proper functionality.…”

“…TRPL::GFP had been utilized in a genetic screen with the chemical mutagen ethyl methane sulfonate (EMS) to uncover components that are involved in membrane protein trafficking. This approach resulted in the isolation of 15 TRPL translocation defective (TTD) mutants and the detailed characterization of the P-Loop-containing, GTP-and phospholipid-binding protein TTD14 [28,33]. Following an RNA-seq screen, another study reported the importance of a CUB and LDLa domain protein (CULD) for endocytic turnover of both Rh1::GFP and TRPL::GFP within Drosophila's PRCs [34].…”