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Maxim Sokolov, Satyabrata Sinha, Marycharmain Belcastro; Fluorescent Labeling of Rod Transducin Subunit in vivo. Invest. Ophthalmol. Vis. Sci. 2012;53(14):756.
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© ARVO (1962-2015); The Authors (2016-present)
To generate a mouse model that allows the subcellular localization of the visual G protein, transducin, to be monitored in real time and in vivo.
A transgenic construct encoding an eGFP-Gγ1 fusion protein was expressed in mouse photoreceptors under the control of a 4.4 kb rhodopsin promoter. The subcellular localization of eGFP-Gγ1 within the rods was determined by fluorescence microscopy. The protein interaction of eGFP-Gγ1 was studied by immunoprecipitation coupled to Western blot analysis.
Transgene-positive mice were characterized by the expression of a 34kD protein specifically recognized by both antibody against Gγ1 and eGFP on Western blots. The expression of this fluorescent fusion protein was limited to rods and mosaic in nature. Green fluorescence was localized to the rod outer segment in dark-adapted mice; however it labeled the entire rod cell in mice exposed to saturating light. When captured by GFP-trap in retinal extracts, this fusion protein co-precipitated together with Gβ1.
Our data demonstrate that transgenic eGFP-tagged Gγ1 readily associates with Gβ1 and undergoes light-driven translocation between the subcellular compartments of rods, which is consistent with the integration of this fusion protein into a functional transducin heterotrimer.
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