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Dusanka Deretic, Jing Wang, Yoshiko Morita; Arf4GTP-ASAP1 Trafficking Module Coordinates VxPx Motif-dependent Ciliary Targeting Of Rhodopsin With Rab11a-Rabin8-Rab8 Ciliogenesis Cascade. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4406.
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We have recently described a ciliary targeting complex that interacts with the VxPx targeting motif of rhodopsin (Mazelova et al., 2009). This complex is comprised of GTP-binding proteins Arf4 and Rab11a, an Arf-GAP ASAP1, and the Arf/Rab11 effector FIP3. In this study we examined the role of ASAP1 in the recruitment of rhodopsin into the ciliary-targeted transport carriers (RTCs) and its interactions with the Rab GTPases involved in ciliary targeting.
Protein complexes generated during rhodopsin trafficking and RTC budding from the Golgi/TGN were examined by immunoprecipitation and GST-pull down. Rhodopsin-eGFP-VxPx construct was expressed in IMCD-3 epithelial cells and the formation of the trafficking complexes was examined as above.
Our data show that the ciliary targeting complex interacts with rhodopsin at the TGN, but not on RTCs. Rhodopsin forms a ternary complex with Arf4GTP and ASAP1 at the TGN. Binding of ASAP1 to rhodopsin involves a secondary ciliary targeting FR motif within the cytoplasmic helix 8, but not the VxPx motif, which is occupied by Arf4 in the ternary complex. ASAP1 binds rhodopsin and FIP3 in a mutually exclusive manner and its interaction with FIP3 my lead to the displacement of rhodopsin from the targeting complex on RTCs. Rhodopsin is correctly targeted to primary cilia of kidney epithelial IMCD cells, in ASAP1-dependent manner. ASAP1 selectively recruits Rab11a and forms a complex with Rab11aGTP, Rab8GDP and its exchange factor Rabin8, providing an activation platform for the regulator of RTC fusion and ciliogenesis, Rab8a. This complex is conserved in ciliated cells.
Our study reveals a novel mechanism for coordination of Arf and Rab GTPases. It indicates an essential role for ASAP1 within the trafficking module that coordinates the ciliary cargo selection with the sequential action of Rab11a-Rabin8-Rab8 ciliogenesis cascade, consequently facilitating compartmentalization of rhodopsin into primary cilia and ROS.
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