Abstract
Activation of phototransduction in the compound eye of Drosophila is mediated by a heterotrimeric G protein that couples to the effector enzyme phospholipase Cbeta. The gamma subunit of this G protein (Ggammae) as well as gamma 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 Ggammae farnesylation, we mutated the farnesylation site and overexpressed the mutated Ggammae in Drosophila. Mass spectrometry of overexpressed Ggammae subunits revealed that nonmutated Ggammae is modified by farnesylation, whereas the mutated Ggammae is not farnesylated. In the transgenic flies, mutated Ggammae forms a dimeric complex with Gbetae, with the consequence that the fraction of non-membrane-bound Gbetagamma is increased. Thus, farnesylation of Ggammae facilitates the membrane attachment of the Gbetagamma complex. We also expressed human Ggammarod in Drosophila photoreceptors. Despite similarities in the primary structure between the transducin gamma subunit and Drosophila Ggammae, we observed no interaction of human Ggammarod with Drosophila Gbetae. This finding indicates that human Ggammarod and Drosophila Ggammae provide different interfaces for the interaction with Gbeta subunits. Electroretinogram recordings revealed a significant loss of light sensitivity in eyes of transgenic flies that express mutated Ggammae. This loss in light sensitivity reveals that post-translational farnesylation is a critical step for the formation of membrane-associated Galphabetagamma required for transmitting light activation from rhodopsin to phospholipase Cbeta.
Highlights
Phototransduction in Drosophila and related flies represents the fastest G protein-coupled signaling system known to date
Characterization of G␥e revealed that it exhibits a carboxylterminal CAAX box (C, cysteine; A, aliphatic amino acid; X, any amino acid, Fig. 1A), which is a common motif of G protein ␥ subunits. ␥ Subunits are prenylated at the conserved cysteine residue of this motif
We show that overexpression of mutated G␥e interferes with membrane attachment of the ␥ complex and that it renders the photoreceptor cells less sensitive to light
Summary
Phototransduction in Drosophila and related flies represents the fastest G protein-coupled signaling system known to date (for reviews, see Refs. 1–5). In the present study we assessed the functional importance of the conserved farnesyl modification of visual G␥ subunits in vivo by generating transgenic flies that express a mutated G␥e without a farnesylation site.
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