June 2021
Volume 62, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2021
Rab11, the TRAPPII complex, and phosphorylation regulate Rabin8 during ciliary trafficking of rhodopsin
Author Affiliations & Notes
  • Dusanka Deretic
    Ophthalmology and Visual Sciences, University of New Mexico Health Sciences Center, Albuquerque, New Mexico, United States
  • Beatrice M Tam
    Ophtalmology, The University of British Columbia, Vancouver, British Columbia, Canada
  • Orson L Moritz
    Ophtalmology, The University of British Columbia, Vancouver, British Columbia, Canada
  • Theresa Fresquez
    Ophthalmology and Visual Sciences, University of New Mexico Health Sciences Center, Albuquerque, New Mexico, United States
  • Footnotes
    Commercial Relationships   Dusanka Deretic, None; Beatrice Tam, None; Orson Moritz, None; Theresa Fresquez, None
  • Footnotes
    Support  RO1 EY 12421
Investigative Ophthalmology & Visual Science June 2021, Vol.62, 535. doi:
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      Dusanka Deretic, Beatrice M Tam, Orson L Moritz, Theresa Fresquez; Rab11, the TRAPPII complex, and phosphorylation regulate Rabin8 during ciliary trafficking of rhodopsin. Invest. Ophthalmol. Vis. Sci. 2021;62(8):535.

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

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Abstract

Purpose : Sorting into rhodopsin transport carriers (RTCs) is regulated by the Rab8 GEF Rabin8, the backbone of the Rab11-Rabin8-Rab8 ciliogenesis complex. Rabin8 phosphorylation by NDR2 (aka STK38L), a canine early retinal degeneration (erd) gene, regulates its membrane association and function. The role of Rabin8 is not clear, however its interactions with multiple ciliary complexes suggest a central role in ciliary pathways of rhodopsin and other sensory receptors.

Methods : A GFP-Rabin8 WT and C (AA 251-460) fragment of human Rabin8 were constructed by site-directed mutagenesis and cloned into the XOP0.8 eGFP-N1 expression vector for the generation of transgenic X. laevis. The following mutants were generated: GFP-Rabin8 S272E and GFP-Rabin8 S272A; GFP-Rabin8 E192A and F201A GEF domain mutants and GFP-Rabin8 Δ300-305 Rab11 binding mutant. The resulting phenotypes were analyzed by confocal microscopy.

Results : GFP-Rabin8 WT co-localized with rhodopsin in the Golgi and on RTCs, similar to the known localization of endogenous Rabin8 in photoreceptors cells. GFP-Rabin8 WT concentrated at the Golgi exit sites (GES), and at the RTC fusion sites. GFP-Rabin8 C was completely cytosolic, indicating that the binding of its N-terminal domain to the TRAPPII complex is essential for localization. GFP-Rabin8 S272E mutant, a phosphomimetic that decreases Rabin8 binding affinity for phosphatidyl serine (PS) but increases affinity for the Sec15 component of the exocyst ciliary complex, was predominantly localized at the base of the cilium, indicating that NDR2 phosphorylation directs Rabin8 to this location. The GFP-Rabin8 S272A mutant accumulated at the GES, where Rabin8 phosphorylation likely occurs. The phenotype of GFP-Rabin8 E192A and F201A mutants, which cannot activate Rab8, was similar to that of the S272E mutant, suggesting that Rabin8 phosphorylation and Rab8 activation occur at the similar site. The GFP-Rabin8 Δ300-305 mutant was completely cytosolic, indicating that Rabin8 binding to Rab11 is also essential for its localization.

Conclusions : Our results implicate Rab11, the TRAPPII complex and the NDR2 kinase in the control of Rabin8 localization and function during the targeting of rhodopsin from the Golgi to the cilium and ROS. Ongoing experiments will establish the linkages between Rabin8 regulatory interactions and the discrete steps in rhodopsin ciliary trafficking.

This is a 2021 ARVO Annual Meeting abstract.

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