Targeted Mutagenesis of the Farnesylation Site ofDrosophilaGγe Disrupts Membrane Association of the G Protein βγ Complex and Affects the Light Sensitivity of the Visual System

Abstract Activation of phototransduction in the compound eye of Drosophila is mediated by a heterotrimeric G protein that couples to the effector enzyme phospholipase Cβ. The γ subunit of this G protein (Gγe) as well as γ subunits of vertebrate transducins contain a carboxyl-terminal CAAX motif (C, cysteine; A, aliphatic amino acid; X, any amino acid) with a consensus sequence for protein farnesylation. To examine the function of Gγe farnesylation, we mutated the farnesylation site and overexpressed the mutated Gγe in Drosophila. Mass spectrometry of overexpressed Gγe subunits revealed that nonmutated Gγe is modified by farnesylation, whereas the mutated Gγe is not farnesylated. In the transgenic flies, mutated Gγe forms a dimeric complex with Gβe, with the consequence that the fraction of non-membrane-bound Gβγ is increased. Thus, farnesylation of Gγe facilitates the membrane attachment of the Gβγ complex. We also expressed human Gγrod in Drosophila photoreceptors. Despite similarities in the primary structure between the transducin γ subunit and Drosophila Gγe, we observed no interaction of human Gγrod with Drosophila Gβe. This finding indicates that human Gγrod and Drosophila Gγe provide different interfaces for the interaction with Gβ subunits. Electroretinogram recordings revealed a significant loss of light sensitivity in eyes of transgenic flies that express mutated Gγe. This loss in light sensitivity reveals that post-translational farnesylation is a critical step for the formation of membrane-associated Gαβγ required for transmitting light activation from rhodopsin to phospholipase Cβ.