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Dusanka Deretic, Beatrice M Tam, Theresa Fresquez, Orson L Moritz; The role of Rabin8 in the ciliary trafficking of rhodopsin. Invest. Ophthalmol. Vis. Sci. 2019;60(9):3979.
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We have previously reported that sorting into rhodopsin transport carriers (RTCs) is regulated by the Arf4-based ciliary trafficking network centered on the scaffold proteins Arf GAP ASAP1, and the Rab8 GEF Rabin8, which organizes the Rab11-Rabin8-Rab8 ciliogenesis cascade, and recruits the R-SNARE VAMP7 onto RTCs to regulate their fusion. NDR2 (also known as STK38L), which was identified as a canine early retinal degeneration (erd) gene corresponding to human ciliopathy Leber congenital amaurosis (LCA), regulates phosphorylation of Rabin8. The role of Rabin8 is not completely clear at present, however its widely recognized interactions with select ciliary proteins, such as TRAPPII complex and the BBSome, suggest a central role in ciliary pathways of sensory receptors.
6His-tagged and GST-tagged human Rabin8 was constructed by subcloning a BamHI-fragment obtained from myc-Rabin8 (a gift from J. Peranen, University of Helsinki, Finland) into pET-28a or pGEX-KG vector. GST-Rabin8 N (AA 1-235) and C (AA 236-460) were constructed by site-directed mutagenesis. A GFP-Rabin8 fusion cDNA was also cloned into the XOP0.8 eGFP-N1 expression vector for the generation of transgenic X. laevis.
GST-pulldowns show that Rabin8 is a part of a complex with Rab11, Rab8 and VAMP7. Pulldowns with GST-Rabin8 N and C indicate that VAMP7 interacts with the Rab11-binding domain of Rabin8. GFP-Rabin8 WT, GFP-tagged Rabin8 N and C, and a NDR2 phosphoryation deficient Rabin8 will be expressed in transgenic X. laevis, and the localization of GFP-tagged proteins detected by immunofluorescence. Interactions of Rabin8-GFP fusion proteins with rhodopsin and all known members of the ciliogenesis cascade, including VAMP7, will be examined by confocal microscopy and Super Resolution (SR) direct Stochastic Optical Reconstruction Microscopy (dSTORM).
Our results implicate Rabin8 in the targeting of rhodopsin from the Golgi to the cilium and ROS. Ongoing experiments will compare the phenotype of GFP-Rabin8 WT with that of Rabin8 mutants in transgenic photoreceptors to determine the mechanisms by which proteins that regulate ciliary trafficking are directly linked to rhodopsin transport mediated by Rabin8.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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