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R. S. Molday, M. C. M. Kwok, L. L. Molday, J. M. Holopainen, L. J. Foster; Proteomics of Photoreceptor Outer Segments Identifies a Subset of SNARE and Rab Proteins Implicated in Membrane Vesicle Trafficking and Fusion. Invest. Ophthalmol. Vis. Sci. 2008;49(13):159. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The goal of this study is to identify most, if not all, proteins present in rod and cone outer segments as a crucial step in understanding the molecular and cellular basis of photoreceptor outer segment structure, function and renewal and identifying candidate proteins associated with inherited retinal degenerative diseases.
Rod outer segments (ROS) were isolated from bovine retina by sucrose gradient centrifugation and further fractionated into soluble and membrane fractions by hypotonic lysis and centrifugation. The ROS membrane fraction was further fractionated into highly pure disk and enriched plasma membrane by immunogold density purturbation centrifugation. All five fractions, ROS, ROS membranes, ROS cytosol, disk membranes and enriched plasma membrane were digested with trypsin following separation by SDS gel electrophoresis. The tryptic peptides were analyzed by high resolution tandem mass spectrometry. Selected proteins implicated in vesicle trafficking and membrane fusion were further analyzed by western blotting and confocal scanning microscopy.
Five hundred and sixteen proteins were identified including 41 proteins that function in rod and cone phototransduction and the visual cycle and most proteins previously shown to be involved in outer segment structure and metabolic pathways. In addition, numerous proteins were detected that have not been previously reported to be present in outer segments including a subset of Rab and SNARE proteins implicated in vesicle trafficking and membrane fusion. Western blotting and immunofluorescence microscopy confirmed the presence of four Rab proteins and a subset of SNARE proteins.
Our proteomic dataset generated by mass spectrometry serves as a valuable resource for further characterizing proteins that play crucial roles in rod and cone outer segment structure, function and renewal. The Rab and SNARE proteins identified and localized in outer segments may play a role in outer segment morphogenesis.
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