Mutation of Light-dependent Phosphorylation Sites of the Drosophila Transient Receptor Potential-like (TRPL) Ion Channel Affects Its Subcellular Localization and Stability

Alexander C Cerny,Tina Oberacker,Jens Pfannstiel,Sebastian Weigold,Carina Will,Armin Huber

doi:10.1074/jbc.m112.426981

Abstract

The Drosophila phototransduction cascade terminates in the opening of the ion channel transient receptor potential (TRP) and TRP-like (TRPL). Contrary to TRP, TRPL undergoes light-dependent subcellular trafficking between rhabdomeric photoreceptor membranes and an intracellular storage compartment, resulting in long term light adaptation. Here, we identified in vivo phosphorylation sites of TRPL that affect TRPL stability and localization. Quantitative mass spectrometry revealed a light-dependent change in the TRPL phosphorylation pattern. Mutation of eight C-terminal phosphorylation sites neither affected multimerization of the channels nor the electrophysiological response of flies expressing the mutated channels. However, these mutations resulted in mislocalization and enhanced degradation of TRPL after prolonged dark-adaptation. Mutation of subsets of the eight C-terminal phosphorylation sites also led to a reduction of TRPL content and partial mislocalization in the dark. This suggests that a light-dependent switch in the phosphorylation pattern of the TRPL channel mediates stable expression of TRPL in the rhabdomeres upon prolonged dark-adaptation.

Highlights

Drosophila TRPL is a cation channel of the phototransduction cascade that undergoes light-dependent subcellular translocation between cell compartments
Drosophila TRPL Is Phosphorylated at Nine Serine and Threonine Residues—We applied mass spectrometry to identify in vivo phosphorylation sites of Drosophila TRPL in dark- and light-adapted flies
The TRPL protein was purified from protein extracts of Drosophila heads by immunoprecipitation and subsequent SDS-gel electrophoresis

Summary

Background

Drosophila TRPL is a cation channel of the phototransduction cascade that undergoes light-dependent subcellular translocation between cell compartments. Mutation of eight C-terminal phosphorylation sites neither affected multimerization of the channels nor the electrophysiological response of flies expressing the mutated channels These mutations resulted in mislocalization and enhanced degradation of TRPL after prolonged dark-adaptation. Mutation of subsets of the eight C-terminal phosphorylation sites led to a reduction of TRPL content and partial mislocalization in the dark This suggests that a light-dependent switch in the phosphorylation pattern of the TRPL channel mediates stable expression of TRPL in the rhabdomeres upon prolonged dark-adaptation. We propose a model in which the subcellular translocation of TRPL is associated with different phosphorylation states that regulate TRPL retention in the rhabdomeres and prevent its degradation

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION