Abstract
Purpose: :
We have recently described a novel ciliary targeting complex that is organized by Arf4, which binds to the VxPx motif present in membrane proteins targeted to primary cilia, including rhodopsin. This trafficking module is comprised of two small GTPases, Arf4 and Rab11, the Rab11/Arf effector FIP3, and the Arf GAP ASAP1 (Mazelova et al., 2009, EMBO J. 28:183). In this study, we examined the spatio-temporal interactions among the components of the complex, and with rhodopsin, during in vitro budding of post-Golgi rhodopsin transport carrier (RTCs).
Methods: :
RTC budding was evaluated using an established cell-free assay. Protein interactions were examined by Co-IPs and GST pulldowns. Transfected IMCD3 cells were examined by confocal microscopy.
Results: :
We found that the recombinant protein containing the BAR, PH, GAP and ankyrin repeat domain of ASAP1 (ASAP1 BAR-PZA), specifically precipitated rhodopsin in the presence of GTP-Arf4, suggesting the formation of a tripartite complex between the Arf, Arf-GAP and rhodopsin prior to RTC budding. We determined that following the GTP-hydrolysis on Arf4, and dissociation from rhodopsin, the ASAP1/Rab11/FIP3 complex remains on RTCs where it may serve as a platform for the successive activation of Rab8, a regulator of RTC-fusion and ciliogenesis. Indeed, we found that ASAP1 forms a complex with GDP-Rab8 and the Rab8 GEF Rabin8 on RTCs. To demonstrate that the ciliary targeting module is conserved in ciliated cells, we employed IMCD3 cells and expressed a fusion protein comprised of bovine rhodopsin and eGFP spliced within the C-terminal tail, leaving the VxPx targeting signal available for molecular interactions. The VxPx motif correctly targeted eGFP-tagged rhodopsin, which co-localized with acetylated α-tubulin in the cilia of IMCD3 cells. In ASAP1 siRNA transfected cells, primary cilia stained with acetylated α -tubulin appeared normal, but lacked the eGFP-tagged rhodopsin. Strikingly, ASAP1 siRNA silenced cells often displayed rhodopsin-eGFP enriched rings around the base of the cilia, which gave rise to actin-rich ectopic protrusions, to which mislocalized rhodopsin was delivered.
Conclusions: :
Our data support the notion that the Arf4/ASAP1/Rab11/FIP3 targeting complex is conserved and involved in the selection and packaging of membrane cargo containing the VxPx motif destined for delivery to the primary cilia in general, and to the ROS in particular.
Keywords: opsins • ciliary processes • protein structure/function