Characterization of two dominant alleles of the major rhodopsin-encoding gene ninaE in Drosophila

PURPOSE In this study we investigated the biochemical and cell biologic characteristics of flies expressing two novel dominant alleles of the major rhodopsin encoding gene neither inactivation nor afterpotential E (ninaE) in a heterozygous background. METHODS Presence of the deep pseudopupil in flies was assayed 5 days post eclosion. For structural analysis, 1-μm-retinal cross sections were obtained from fixed and resin-embedded Drosophila heads. Confocal microscopy was performed on dissected retinas stained with antibodies specific for rhodopsin, NinaA, and F-actin. Rhodopsin levels were determined by western and slot blot analysis. RESULTS Dominant rhodopsin mutants showed progressive age-dependent and light-independent loss of the deep pseudopupil, without any apparent retinal degeneration at the morphological level. Expression of mutant rhodopsin caused rhodopsin to mislocalize to the cell body and the endoplasmic reticulum compartment. Mutant rhodopsin also caused loss of solubility of wild-type rhodopsin and its accumulation presumably as a high molecular mass complex in the photoreceptor cell body. CONCLUSIONS In heterozygous mutant flies, there is loss of wild-type rhodopsin immunoreactivity on a western assay but less reduction using slot blot analysis. This suggests that mutant rhodopsin is likely inducing the misfolding and insolubility of wild-type rhodopsin. Localization of rhodopsin revealed that in mutant flies, wild-type rhodopsin is mislocalized to the cell body and the endoplasmic reticulum.