Abstract
Purpose :
The identity of the R-SNARE involved in the fusion of rhodopsin transport carriers (RTC) that pairs with Qabc-SNAREs Syntaxin 3 and SNAP-25 on the rod inner segment plasma membrane to deliver rhodopsin to the outer segment of rod photoreceptors is unknown. We have tested vesicle-associated membrane protein 7 (VAMP7) as a candidate R-SNARE. VAMP7 consists of a regulatory longin domain (LD), a SNARE motif and a transmembrane domain. LD is phosphorylated at Tyr-45 by c-Src kinase, which activates both SNARE binding and exocytosis.
Methods :
Immunoprecipitation (IP) using post-nuclear supernatant from frog retinas was performed. Multiple constructs of VAMP7-GFP fusion proteins were expressed in Xenopus laevis photoreceptors under the control of Xenopus rhodopsin promoter. A combination of proximity ligation assay (PLA), immunostaining and in vitro biochemical interaction studies were used to identify the localization of VAMP7 and its potential interacting partners.
Results :
We have examined VAMP7 regulation during formation, trafficking and targeting of RTCs to the fusion site. Based on the IP data, VAMP7 forms a complex with Syntaxin 3 and SNAP-25. Immunostaining and PLA show that VAMP7 co-localizes with Syntaxin 3 at the RTC fusion site. Based on immunostaining, VAMP7 also co-localizes with rhodopsin, both in the Golgi/TGN and on RTCs. The localization of the phosphomimetic GFP-VAMP7 (Y45E) and GFP-VAMP7-ΔLD in transgenic Xenopus are similar to that of endogenous VAMP7, whereas the non-phosphorylatable GFP-VAMP7 (Y45F) mutant is retained in the Golgi. The GFP-VAMP7 (R150E) with a mutation in the SNARE motif shows aberrant localization in or around the Golgi while the double mutant (Y45E/R150E) localizes predominantly to the plasma membrane. Based on protein interaction and PLA assays, VAMP7 interacts with Rab11, Rabin8 and Rab8, which form the RTC targeting complex.
Conclusions :
The ability of VAMP7 to form a complex with Syntaxin 3 and SNAP-25, along with its localization on RTCs at the fusion site, strongly suggest that VAMP7 is the R-SNARE involved RTC fusion. The Tyr-45 phosphorylation plays a critical role in the subcellular localization of VAMP7. The R150E mutation in the SNARE motif appears to preclude its interaction with the components of the Rabin8-Rab11-Rab8 targeting complex.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.