Abstract
The Arf4-rhodopsin complex (mediated by the VxPx motif in rhodopsin) initiates expansion of vertebrate rod photoreceptor cilia-derived light-sensing organelles through stepwise assembly of a conserved trafficking network. Here, we examine its role in the sorting of VAMP7 (also known as TI-VAMP) - an R-SNARE possessing a regulatory longin domain (LD) - into rhodopsin transport carriers (RTCs). During RTC formation and trafficking, VAMP7 colocalizes with the ciliary cargo rhodopsin and interacts with the Rab11-Rabin8-Rab8 trafficking module. Rab11 and Rab8 bind the VAMP7 LD, whereas Rabin8 (also known as RAB3IP) interacts with the SNARE domain. The Arf/Rab11 effector FIP3 (also known as RAB11FIP3) regulates VAMP7 access to Rab11. At the ciliary base, VAMP7 forms a complex with the cognate SNAREs syntaxin 3 and SNAP-25. When expressed in transgenic animals, a GFP-VAMP7ΔLD fusion protein and a Y45E phosphomimetic mutant colocalize with endogenous VAMP7. The GFP-VAMP7-R150E mutant displays considerable localization defects that imply an important role of the R-SNARE motif in intracellular trafficking, rather than cognate SNARE pairing. Our study defines the link between VAMP7 and the ciliary targeting nexus that is conserved across diverse cell types, and contributes to general understanding of how functional Arf and Rab networks assemble SNAREs in membrane trafficking.