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Vasundhara Kandachar, Beatrice M Tam, Orson L Moritz, Dusanka Deretic; VAMP7 as a regulator of rhodopsin transport carrier fusion in rod photoreceptors. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1731.
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© ARVO (1962-2015); The Authors (2016-present)
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.
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.
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.
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.
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