Author/Editor		Schillo, Simone; Belušič, Gregor; Hartmann, Kristina; Franz, Claudia; Kuehl, Boris; Brenner-Weiss, Gerald; Paulsen, Reinhard; Huber, Armin
Title		Targeted mutagenesis of the farnesylation site of Drosophila Ggammae discrupts membrane association of the G protein betagamma complex and affects the light sensitivity of the visual system
Type		članek
Source		J Biol Chem
Vol. and No.		Letnik 279, št. 35
Publication year		2004
Volume		str. 36309-16
Language		eng
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 (Gye) 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 funetion of Ggammae farnesylation, we mutated the farnesylation site and overexpressed the mutated Ggammae in Drosophala. 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 Gbetay is inereased. 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 interfaees for the interaction with Gbeta subunite. 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.
Descriptors		DROSOPHILA PHOTOTRANSDUCTION GTP-BINDING PROTEINS PROTEIN ISOPRENYLATION PHOTORECEPTORS, INVERTEBRATE TRANSDUCIN SPECTRUM ANALYSIS, MASS ELECTRORETINOGRAPHY MUTAGENESIS, SITE-DIRECTED