title: Targeted mutagenesis of the farnesylation site of Drosophila Ggammae disrupts membrane association of the G protein betagamma 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 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.
authors.affiliation: Institut für Zoologie, Universität Karlsruhe, Haid-und-Neu-Strasse 9, Karlsruhe 76131, Germany.
authors.forename: Simone
authors.initials: S
authors.lastname: Schillo
authors.forename: Gregor
authors.initials: G
authors.lastname: Belusic
authors.forename: Kristina
authors.initials: K
authors.lastname: Hartmann
authors.forename: Claudia
authors.initials: C
authors.lastname: Franz
authors.forename: Boris
authors.initials: B
authors.lastname: Kühl
authors.forename: Gerald
authors.initials: G
authors.lastname: Brenner-Weiss
authors.forename: Reinhard
authors.initials: R
authors.lastname: Paulsen
authors.forename: Armin
authors.initials: A
authors.lastname: Huber
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D030801:Animals, Genetically Modified; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D003459:Crystallins / Q000235:genetics / Q000502:physiology*; D003545:Cysteine / Q000737:chemistry; D004247:DNA / Q000378:metabolism; D004330:Drosophila; D004591:Electrophoresis, Polyacrylamide Gel; D004596:Electroretinography; D020962:Heterotrimeric GTP-Binding Proteins / Q000502:physiology; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000378:metabolism; D008027:Light; D013058:Mass Spectrometry; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D054799:Phospholipase C beta; D020795:Photophobia; D017956:Photoreceptor Cells, Invertebrate / Q000378:metabolism / Q000502:physiology*; D017354:Point Mutation; D011233:Precipitin Tests; D017368:Protein Prenylation; D011499:Protein Processing, Post-Translational; D012160:Retina / Q000378:metabolism; D012243:Rhodopsin / Q000737:chemistry; D019076:Transgenes; D010738:Type C Phospholipases / Q000378:metabolism; D038222:gamma-Crystallins
chemical_list: D003459:Crystallins; D007527:Isoenzymes; C107256:crystallin gammaE; D038222:gamma-Crystallins; D004247:DNA; D012243:Rhodopsin; D010738:Type C Phospholipases; D054799:Phospholipase C beta; D020962:Heterotrimeric GTP-Binding Proteins; D003545:Cysteine