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Vasundhara Kandachar, Beatrice M Tam, Orson L Moritz, Dusanka Deretic; Fine-mapping of the interaction between R-SNARE VAMP7 and ciliary targeting complex in rod photoreceptors. Invest. Ophthalmol. Vis. Sci. 2017;58(8):634.
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© ARVO (1962-2015); The Authors (2016-present)
The regulation of the uptake of R-SNARE VAMP7 into the RTCs targeted to the rod inner segment (RIS) plasma membrane to mediate the fusion of rhodopsin transport carriers (RTC) that deliver rhodopsin to the rod outer segment is unknown. The RTCs are directed to the RIS plasma membrane by the ciliary targeting complex comprising Rab11a, Rabin8 and Rab8 proteins. We have fine-mapped the interaction of VAMP7 with the components of the ciliary targeting complex.
Immunoprecipitation (IP) and GST pull-down experiments using post-nuclear supernatant from frog retinas were performed. Multiple constructs of GFP-VAMP7 fusion proteins were expressed in Xenopus laevis photoreceptors under the control of Xenopus rhodopsin promoter. Correlative light and electron microscopy (CLEM) was used to examine Xenopus retina expressing GFP-VAMP7-R150E. A combination of proximity ligation assay (PLA) and in vitro protein interaction studies were used to identify the localization of VAMP7 and its interacting partners.
VAMP7 consists of a regulatory longin domain (LD), a SNARE domain (SD) and a transmembrane domain. Our protein interaction studies revealed that VAMP7 binds to Rabin8 via its SD while it binds to activated Rab11-GTP and inactive Rab8-GDP through its LD. These interactions were observed in the Golgi on RTCs and the interaction with Rab8 was additionally seen in the proximity of RIS plasma membrane, as determined by PLA. Our interaction studies also revealed that VAMP7 does not bind to Rab11a while Rab11a is bound to its effector FIP3. CLEM analysis of Xenopus retina expressing GFP-VAMP7-R150E with the mutation in SD showed intracellular accumulation of the mutant protein in vesicular structures closely apposed to the mitochondria and accumulation of lysosomes. While this accumulation caused disruption of cellular architecture, photoreceptor cell death and retinal degeneration were not observed.
VAMP7 interacts with Rabs through its LD and with Rabin8 via its SD. VAMP7 binding to Rabin8 and Rab11a is reminiscent of the Rab11a-FIP3-Rabin8 dual effector complex with FIP3 replaced by VAMP7. The accumulation of the mutant GFP-VAMP7-R150E in intracellular membranes suggests that the SNARE motif is involved in ciliary targeting.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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