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D. Deretic, J. Mazelova, B. Tam, O. Moritz, P. Randazzo; Rhodopsin Ciliary Targeting VxPx Motif Couples Trafficking With Photoreceptor Morphogenesis Through an Arf4-Based Module. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1684.
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© ARVO (1962-2015); The Authors (2016-present)
Direct binding of rhodopsin C-terminal VxPx ciliary targeting signal to the small GTP-binding protein Arf4 initiates the assembly of a trafficking protein complex that includes the Arf-GAP ASAP1. In this study we examined the effects of an Arf4 mutant deficient in ASAP1-mediated GTP hydrolysis on rhodopsin trafficking and photoreceptor morphology.
Transgenic X. laevis expressing Arf4-GFP fusion proteins were generated, identified by G418 resistance and examined by confocal microscopy. Arf-GAP activity was determined using a fixed time point assay in which the hydrolysis of [α32P]GTP bound to Arf4 was measured.
Previous analysis revealed that the [I46D]Arf1 mutation in the Switch 1 domain selectively reduced ASAP1-induced GTP hydrolysis. We introduced the I46D mutation into Arf4 and determined that this mutation also specifically affected ASAP1-mediated GTP hydrolysis. We generated transgenic X. laevis expressing Arf4-GFP and [I46D]Arf4-GFP fusion proteins in their rod photoreceptors. While Arf4-GFP had no discernable effect on the transgenic retinas, [I46D]Arf4-GFP caused robust retinal degeneration indicating a dominant negative effect of the mutant on endogenous Arf4. The Golgi appeared disorganized and RIS were often constricted around the Golgi in photoreceptors expressing [I46D]Arf4-GFP. Phalloidin staining revealed a significant perturbation of the actin cytoskeleton in these retinas, which included a substantial constriction of adherens junctions to the point of division of the RIS. The mutant Arf4-GFP also affected rhodopsin trafficking. Substantially enlarged rhodopsin-positive budding profiles emanated from the Golgi, occasionally directed away from the ROS, towards the synaptic pole of the cell.
Our data show that Arf4 plays an essential role in rhodopsin trafficking from the Golgi to the ROS in vivo. An Arf4 mutant impaired in ASAP1-mediated GTP hydrolysis affects the function of the ciliary targeting complex and causes trafficking, cytoskeletal and morphological defects resulting in retinal degeneration. Our findings define a novel mechanism for the coupling of membrane trafficking and photoreceptor morphogenesis.
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