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Proteomic analysis of rod photoreceptor outer segment and characterization of an aminophospholipid transporter-like ATPase Kwok, Michael Chung Ming.

Abstract

The photoreceptor outer segment is a specialized compartment of rod and cone photoreceptor cells which functions in vertebrate phototransduction, the process which converts light into electrical signals as the initial step in vision. Although the molecular mechanisms underlying phototransduction and light adaptation have been well examined, other cellular and molecular processes in the photoreceptor outer segment are not clearly understood. These include the mechanism of outer segment renewal, in particular the morphogenesis and shedding of disk membranes at opposite ends of the outer segment. In this study, a mass spectrometry-based proteomic approach was used to identify the complete proteome of the rod photoreceptor outer segment. Rod outer segments were isolated from bovine retina and separated into different subcellular preparations, followed by analysis on a tandem mass spectrometer. A total of 529 proteins were identified, including a large number of proteins that have not been previously reported to be present in the outer segments including a subset of proteins implicated in vesicle trafficking and membrane fusion. Using western blotting and immunofluorescence techniques, it was confirmed that the small GTPase Rab 11 and the Rab accessory protein Rab-GDI, as well as the SNARE protein VAMP 2/3 and SNARE-associated protein Munc 18-1 are true components of the rod outer segment. In addition, several uncharacterized proteins were identified one of which is aminophospholipid transporter-like ATPase IB (ATP8A2). The gene was cloned, expressed in cultured cells, and immunoaffinity purified. ATPase activity assay using radiolabeled [α-³²P] ATP showed that the purified protein was highly activated in the presence of brain polar lipid extract. Immunocytochemical studies showed a colocalization of the expressed protein with the ER-resident protein calnexin when expressed in HEK293 cells. These results, along with the proteomic data suggest that ATPase IB is a membrane protein in the rod outer segment that may play a role in transporting aminophospholipid across the membrane bilayer using ATP as an energy source. This study serves as a basis for understanding the role of this previously uncharacterized protein in the rod photoreceptor and determining whether mutations in the ATP8A2 gene may cause retinal disorders.

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