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Wolfgang Baehr, Cecilia D gerstner, Christin Hanke-Gogokhia, Guoxin ying, Jeanne M Frederick; Trafficking of transducin-α by UNC119A/B and ARL3GTP dependent diffusion. Invest. Ophthalmol. Vis. Sci. 201657(12):.
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© ARVO (1962-2015); The Authors (2016-present)
UNC119A and UNC119 B are closely related acyl-binding proteins expressed in photoreceptors. UNC119A forms an immunoglobulin-like β-sandwich fold into which the N-terminal acyl side chain of Tα inserts. Our purpose was to generate UNC119A/B double knockouts and explore the consequences on transducin trafficking.
GST-pull downs, preparation of a monospecific antibody, western blotting, Unc119 double knockout mouse, immunohistochemistry.
UNC119A and UNC119B are expressed ubiquitously among vertebrates. In the mammalian eye, UNC119s are strongly expressed in photoreceptors. Amino acid sequence comparison of both UNC119 isoforms reveal perfect conservation of all amino acids known to interact with the acyl side chain and N-terminal residues of Tα, and predict nearly identical structures. Both GST-UNC119B and GST-UNC119/RG4 pull down Tα from retina lysates, suggesting closely related functions as acyl-binding proteins. We generated Unc119a/b double knockout mice by breeding doubly heterozygous parents (Unc119a+/-;Unc119b+/-). Double knockout mice are viable, fertile and stable. Tα is present in dark-adapted Unc119 dKO rod outer segments, but mislocalized in the myoid region of the Unc119 dKO inner segment and ONL. These results support the idea that UNC119 polypeptides traffic Tα by diffusion in the dark-adapted rod. Trafficking by diffusion is further supported by dependence of Tα delivery to the outer segments by ARL3GTP, acting as a cargo dislocation factor. A model of UNC119/ARL3-dependent trafficking will be presented. Preliminary results show that the Unc119 dKO a-wave is attenuated at 6 weeks of age. A slowly developing retina degeneration is complete (single row of ONL nuclei) in the dKO at 11 months of age, while the Unc119a+/-;Unc119b+/- heterozygote littermate appears normal.
It is unclear how transducin traffics to the distal RIS and to the ROS. Our results suggest that transducin traffics in dark-adapted rods to the OS at least in part by UNC199A/B-dependent diffusion.
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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