Abstract
Purpose :
The Rab11-Rabin8-Rab8 ciliogenesis complex regulates the formation of rhodopsin transport carriers (RTCs). The Rab8 GEF Rabin8 is phosphorylated at S272 by NDR2 kinase (aka STK38L), a canine early retinal degeneration (erd) gene. NDR2 phosphorylation decreases Rabin8 binding to phosphatidyl serine (PS) in the Golgi while increasing affinity for the Sec6/8 complex (exocyst) at the cilium. In this study, we sought to define the step at which NDR2 phosphorylation of Rabin8 regulates the Rab11-Rab8 succession in the ciliary trafficking of rhodopsin.
Methods :
We generated transgenic X. laevis expressing WT or the following mutants of human GFP-Rabin8: phosphomimetic S272E, the nonphosphorylatable S272A; the Rab8 GEF mutants E192A and F201A; the Rab11 binding mutant T419A Y423A L428A; and the Rab8 GEF mutant/non-phosphorylatable F201A S272A, and analyzed them by confocal, Airyscan, and 3D Structured Illumination microscopy (SIM). Interactions of GST-Rabin8 mutants with Rab8 and Rab11 were examined by GST pulldowns.
Results :
The human GFP-Rabin8 WT colocalized in transgenic rods with endogenous Rabin8 and rhodopsin in the Golgi and on RTCs. Possibly saturating endogenous NDR2, it accumulated at the Golgi exit sites (GES) along with the endogenous Rabin8. E192A and F201A mutants colocalized with rhodopsin in the Golgi, at the GES, and on substantially enlarged RTCs, likely exerting the dominant negative effect on Rab8 function. GST pulldowns showed direct interactions with Rab8 and Rab11. T419A Y423A L428A mutant was cytosolic indicating that Rab11 is essential for Rabin8 membrane localization. The S272E phosphomimetic colocalized with endogenous Rabin8 at the GES and on RTCs, whereas the S272A and the F201A S272A mutant principally accumulated at the GES. Airyscan and 3D SIM analysis revealed that GES appear as tubulo-vesicular membranous aggregates, probably corresponding to a distended trans-Golgi network (TGN), which in S272A-expressing photoreceptors abnormally breaks up into large fragments that occasionally advance toward the cilium.
Conclusions :
Our study implicates the GES/TGN as a checkpoint where NDR2 kinase phosphorylates Rabin8, allowing normal membrane progression to the cilium. It shows that Rabin8 phosphorylation at the GES/TGN precedes Rab8 activation on RTCs. Disturbance of these well-ordered processes likely underlies the retinal degeneration caused by disruption of NDR2/STK38L.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.