ABSTRACT
Light stimulates phosphatidylinositol bisphosphate phospholipase C (PLC) activity in Drosophila photoreceptors. We have investigated the mechanism of this reaction by assaying PLC activity in Drosophila head membranes using exogenous phospholipid substrates. PLC activation depends on the photoconversion of rhodopsin to metarhodopsin and is reduced in norpAEE5 PLC and ninaEP332 rhodopsin mutants. NorpA PLC is stimulated by light at free Ca2+ concentrations between 10 nM and 1 microM. This finding is consistent with a Ca(2+)-mediated positive feedback mechanism that contributes to the rapid temporal response of invertebrate photoreceptor cells. The guanyl nucleotide dependence of light-stimulated PLC activity indicates that a G protein regulates NorpA. This was confirmed by the observation that light stimulation of PLC activity is deficient in mutants that lack the eye-specific G protein beta subunit G beta e. These results indicate that G beta e functions as the beta subunit of the G protein coupling rhodopsin to NorpA PLC.
Subject(s)
Drosophila Proteins , Drosophila/physiology , GTP-Binding Proteins/metabolism , Phosphoric Diester Hydrolases/metabolism , Photoreceptor Cells, Invertebrate/physiology , Type C Phospholipases , Animals , Calcium/pharmacology , Dose-Response Relationship, Drug , Dose-Response Relationship, Radiation , Drosophila/enzymology , Drosophila/radiation effects , Enzyme Activation/radiation effects , Feedback , GTP-Binding Proteins/genetics , Head/physiology , Light , Membranes/physiology , Phosphatidylinositol Diacylglycerol-Lyase , Phospholipase C beta , Photoreceptor Cells, Invertebrate/enzymologyABSTRACT
An in situ 35S-labeled guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) binding procedure was developed to assay light-stimulated G protein activity in Drosophila compound eyes. We found that Drosophila with mutations in G beta e, an abundant photoreceptor-specific G protein beta subunit essential for photoexcitation, are defective in light-stimulated [35S]GTP gamma S binding. We confirmed that G beta e interacts with a GTP-binding protein by demonstrating that immunoprecipitation of G beta e is sensitive to GTP gamma S. These results suggest that G beta e functions as the beta subunit of a heterotrimeric G protein that couples photoactivation of rhodopsin to downstream components in the Drosophila phototransduction cascade.
