Abstract
Purpose: :
A ciliary targeting complex comprised of GTP-binding proteins Arf4 and Rab11a, an Arf-GAP ASAP1, and the Arf/Rab11 effector FIP3 regulates ciliary targeting of rhodopsin (Mazelova et al., 2009). In this study we examined the role of ASAP1 in organizing the events that link rhodopsin's recognition to its ciliary and ROS destination.
Methods: :
Wild type and mutant rhodopsin-eGFP-VxPx constructs were expressed in IMCD-3 epithelial cells and the delivery to the cilia was examined by confocal microscopy. Protein complexes generated during rhodopsin trafficking in photoreceptor cells were analyzed by immunoprecipitation and pull down experiments.
Results: :
Our data show that the initial event in rhodopsin sorting at the photoreceptor Golgi/TGN is its formation of a tripartite complex with Arf4GTP and the Arf-GAP ASAP1, which recognize the VxPx and the FR ciliary targeting motifs of rhodopsin, respectively. We find that rhodopsin binds to the BAR domain of ASAP1, responsible for its oligomerization and membrane curvature-inducing activity. The Arf/Rab11 effector FIP3, which also binds the BAR domain and stimulates the Arf GAP activity of ASAP1, substantially competes the binding of 60 nM rhodopsin to ASAP1. Similarly, rhodopsin competes the binding of 15 nM FIP3 to ASAP1, suggesting that the binding sites of rhodopsin and FIP3 on ASAP1 are at least partially overlapping. FIP3 displaces rhodopsin from the tripartite complex with ASAP1 and Arf4GTP, but has negligible effect on the binding of 180 nM Arf4GTP to ASAP1. Therefore, rhodopsin displacement is likely coupled to FIP3-stimulated GTP hydrolysis on Arf4 by ASAP1. We find that ASAP1 next serves as a platform that brings together the proteins necessary for transport to the cilia including the two G-proteins of the Rab family-- Rab11a and Rab8--linked by the Rab8 guanine nucleotide exchange factor Rabin8. Ciliary localization of rhodopsin-GFP-VxPx expressed in epithelial cells is lost upon siRNA-mediated knockdown of ASAP1. Mutant [FR-AA]rhodopsin-GFP-VxPx, defective in ASAP1 binding, does not engage Rab8, fails to translocate across the periciliary diffusion barrier and reach the cilia.
Conclusions: :
Our study identifies the Arf/Rab11 effector FIP3 as a candidate factor for the displacement of rhodopsin from ASAP1-associated complexes that control its targeting. In addition, we show the essential role of ASAP1 in linking ciliary cargo, such as rhodopsin, with the Rab11a-Rabin8-Rab8 ciliogenesis module that also controls the ability of rhodopsin to enter the primary cilia and, consequentially, the ROS.
Keywords: opsins • photoreceptors • protein structure/function