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Bridgette Ray, Beatrice Tam, Orson Moritz, Dusanka Deretic; VAMP7 as a Regulator of Rhodopsin Transport Carrier Fusion in Photoreceptors. Invest. Ophthalmol. Vis. Sci. 2013;54(15):706.
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Our lab has previously identified Syntaxin3 and SNAP25 as the Qa and Qbc-SNAREs that are present on the rod inner segment (RIS) plasma membrane. We have also shown that these SNAREs are involved in the regulation of Rhodopsin transport carrier (RTC) fusion (Mazelova et al, 2009). However, the R-SNARE present on the RTCs that interacts with Syntaxin3 and SNAP25 on the RIS membrane has not been identified. In this study, we identified VAMP7 as a candidate for the R-SNARE present on RTCs.
Post-nuclear supernatant (PNS) was isolated from frog retinas and fractionated on sucrose density gradients. VAMP7 localization in control and propranolol treated retinas was studied using confocal microscopy. Full length wild type VAMP7 or an N-terminal fragment of VAMP7, which acts as a dominant negative, will be expressed specifically in the photoreceptors of X. laevis. The retinas of the transgenic frogs will be analyzed by confocal microscopy to determine the effects of VAMP7 on the delivery of rhodopsin to the ROS.
Our data show that VAMP7 is present in both the RTCs and Golgi/TGN. Interestingly, VAMP7 co-fractionates with ASAP1 in both the Golgi/TGN and RTC fractions. Confocal microscopy reveals the presence of VAMP7 within the RIS of frog photoreceptors. When the retinas were treated with propranolol, which causes membrane-tethering defects and inhibits RTC fusion, there was a marked accumulation of VAMP7 on the RIS membrane.
Our study suggests that VAMP7 is present on RTCs. When retinas were treated with propranolol, VAMP7 accumulated on the RIS membrane in a manner similar to what has been reported for SNAP25 and Syntaxin3, suggesting an interaction (Mazelova, 2009). Taken together, our data suggests that VAMP7 might be the R-SNARE that interacts with Syntaxin3 and SNAP 25, resulting in fusion of RTCs with the RIS membrane.
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