Abstract
Purpose: :
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.
Methods: :
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.
Results: :
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.
Conclusions: :
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.
Keywords: photoreceptors • retinal degenerations: cell biology • transgenics/knock-outs