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Dusanka Deretic, Bea Tam, Orson L Moritz, Vasundhara Kandachar; R-SNARE VAMP7 in rhodopsin trafficking and phototransduction membrane renewal. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4977. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
We have previously reported that sorting and uptake of the R-SNARE vesicle-associated membrane protein 7 (VAMP7) into rhodopsin transport carriers (RTCs) are regulated by the hitherto identified Arf4-based ciliary trafficking network. Analysis of transgenic Xenopus photoreceptors expressing GFP-VAMP7-R150E, with the mutation in the zero layer of the SNARE motif—thought to be essential for SNARE paring and membrane fusion—showed profoundly disrupted cellular architecture with accumulation of aberrant vesicular structures closely apposed to the Golgi. In this study we examined the nature and composition of these membranes using proteomic analysis.
GFP-VAMP7-R150E, and the non-phosphorylatable (inactive) GFP-VAMP7-Y45F mutant that accumulates in the Golgi, were expressed in Xenopus laevis photoreceptors under the control of Xenopus rhodopsin promoter. Retinas were isolated from F1 adults and subjected to immuoprecipitation using anti-GFP antibody. Precipitated proteins were analyzed by LC-MS/MS.
Substantial differences were noted in the precipitated proteins associated with the two VAMP7 mutants. Particularly, a number of actin-cytoskeleton related proteins, including unconventional Myosin 6, involved in membrane trafficking from the Golgi, and Arp2/3 complex, involved in actin nucleation, were associated with the Y45F mutant, but nearly or completely absent from the R150E mutant. By contrast, membranes harboring R150E mutant contained a significant number of ROS phototransduction proteins. Three-fold enrichment in rhodopsin was observed, but the most dramatic differences were found in ABCA4 transporter, arrestin and peripherin-2/RDS, all of which were absent from Y45F sample. GFP-VAMP7-R150E precipitate was also enriched in transducin α and β subunits, PDE6β, rhodopsin kinase (GRK1), recoverin and the cyclic nucleotide-gated channel α subunit.
Both the absence of interaction with known cytoskeletal proteins involved in membrane budding as well as the accumulation of membranes containing ROS proteins in mutant R150E expressing rods implicates VAMP7 in the targeting of rhodopsin and associated phototransduction proteins from the Golgi to the cilium and ROS. Ongoing refinement and comparison with the VAMP7 WT will inform further classification of phototransduction proteins whose transport to the ROS is linked to rhodopsin and mediated by VAMP7.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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