September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
VAMP7 as a regulator of rhodopsin transport carrier fusion in rod photoreceptors
Author Affiliations & Notes
  • Vasundhara Kandachar
    Department of Surgery/Division of Ophthalmology, University of New Mexico School of Medicine, Albuquerque, New Mexico, United States
  • Beatrice M Tam
    Department of Ophthalmology and Visual Sciences, University of British Columbia, Vancouver, British Columbia, Canada
  • Orson L Moritz
    Department of Ophthalmology and Visual Sciences, University of British Columbia, Vancouver, British Columbia, Canada
  • Dusanka Deretic
    Department of Surgery/Division of Ophthalmology, University of New Mexico School of Medicine, Albuquerque, New Mexico, United States
  • Footnotes
    Commercial Relationships   Vasundhara Kandachar, None; Beatrice Tam, None; Orson Moritz, None; Dusanka Deretic, None
  • Footnotes
    Support  EY12421, CIHR-MOP64400, NSERC, Foundation Fighting Blindness-Canada
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 1731. doi:
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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)

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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.

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