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Maxim Sokolov, Marycharmain Belcastro, Joseph Murphy, Saravanan Kolandaivelu; Posttranslational Lipid Modification Controls the Trafficking of Transducin to the Photoreceptor Cilium. Invest. Ophthalmol. Vis. Sci. 201657(12):.
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© 2017 Association for Research in Vision and Ophthalmology.
The signaling functions of various cilia are predicated based on the trafficking of heterotrimeric G proteins into this organelle, however, the mechanism underlying these processes remains poorly understood. This is exemplified in rod photoreceptors, whose sensory cilia, termed the outer segments, detect photons using the G protein, transducin. Like all G proteins, the transducin Gγ1 subunit undergoes a posttranslational lipid modification. Here, we tested the role of this farnesyl lipid group in the trafficking of transducin to the rod outer segment.
Epitope-tagged Gγ1, carrying a Cys71Ser substitution, which eliminates its farnesylation site (HA-Gγ1-no-farnesyl), was expressed in the photoreceptors of transgenic mice. An identical construct without the mutation (HA-Gγ1-wild type) was used as a control. The protein interactions of HA-Gγ1-no-farnesyl and HA-Gγ1-wild type were analyzed by pull down assay and Western blotting, and their subcellular localization was monitored by immunofluorescence confocal microscopy.
Both HA-Gγ1-no-farnesyl and HA-Gγ1-wild type were clearly detectable in rods, while their overall levels in the retina were only about 1% that of endogenous Gγ1. No detrimental effects of the transgenes on photoreceptors were noticed. HA-Gγ1-no-farnesyl and HA-Gγ1-wild type both formed a complex with the endogenous Gβ1. The subcellular localization of the resulting Gβ1γ1 complexes was, however, very different. While HA-Gβ1γ1-no-farnesyl was strictly excluded from the rod outer segments, HA-Gβ1γ1-wild type exhibited a very pronounced translocation between the outer segments and the rod inner segments and synapses that was dependent on the history of light stimulation.
Our data demonstrate that the farnesylation of Gγ1 controls the trafficking of transducin to the photoreceptor cilia, while being nonessential for Gβ1γ1 complex assembly. The mechanism consistent with this observation will be discussed.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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