The Cystein-Rich Domains of RDGA, a Drosophila Eye-Specific Diacylglycerol Kinase, Are Required for Protein Localization and Enzyme Activity

Abstract

Retinal DeGeneration A (RDGA) is a Drosophila eye-specific diacylglycerol kinase (DGK), whose genetic mutation blocks photoreceptor potentials and causes severe retinal degeneration. Drosophila phototransduction is mediated by the phosphoinositide (PI) signaling, initiated by the G-protein coupled phospholipase C (NORPA). Within this cascade, RDGA mediates an initial step for the regeneration of PIs by the production of phosphatidic acid, and attenuates the intracellular level of DG that regulates the activity of the light-sensitive membrane channels (TRP channels). In a photoreceptor cell, RDGA protein localizes to the restricted subcellular region called the subrhabdomeric (SR) region that is adjacent to the photoreceptive organelle, the rhabdomere. This region contains a specialized network of smooth endoplasmic reticulum, subrhabdomeric cistern (SRC), and RDGA is thought to function in association with the membranes of SRC. Thus, the localization of RDGA to the SR region appears to be important for its function. We have asked which domain of RDGA protein is necessary for its subcellular localization and function. The structure of RDGA protein predicted from the gene contains tandemly arranged cystein-rich domains (CRDs) in the N-terminal region, a catalytic domain in the middle region, and four ankyrin-like repeats at the C-terminus The rescue experiments by germ-line transformation on rdgA null mutants expressing RDGA proteins that are deleted in these domains showed that the CRDs are necessary for the intracellular localization and function of RDGA. The RDGA protein without these domains does not rescue the degeneration phenotype, shows no recovery of DGK activity, and mislocalizes in the cytoplasm of photoreceptors. This is, to our knowledge, the first demonstration of the function of CRDs of DGKs in vivo.