May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
Rhodopsin Ciliary Targeting VxPx Motif Couples Trafficking With Photoreceptor Morphogenesis Through an Arf4-Based Module
Author Affiliations & Notes
  • D. Deretic
    Surgery, Univ of New Mexico Sch of Med, Albuquerque, New Mexico
  • J. Mazelova
    Surgery, Univ of New Mexico Sch of Med, Albuquerque, New Mexico
  • B. Tam
    Ophthalmology, UBC, Vancouver, British Columbia, Canada
  • O. Moritz
    Ophthalmology, UBC, Vancouver, British Columbia, Canada
  • P. Randazzo
    NCI, NIH, Bethesda, Maryland
  • Footnotes
    Commercial Relationships  D. Deretic, None; J. Mazelova, None; B. Tam, None; O. Moritz, None; P. Randazzo, None.
  • Footnotes
    Support  EY 12421
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 1684. doi:https://doi.org/
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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. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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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 
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