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A. F. Goldberg, B. Tam, L. M. Ritter, N. Khattree, O. L. Moritz, S. K. Chintala, L. Dang; Visualization of GARP-Mediated Protein-Protein Interactions Governing Photoreceptor Outer Segment Architecture. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5458.
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© ARVO (1962-2015); The Authors (2016-present)
Vertebrate photoreceptors respond to light via a G-protein mediated cascade organized within a highly membranous outer segment (OS). The interactions that establish and regulate the dynamic OS architecture are critical for photoreceptor function and viability, but are not well understood. We sought to develop a versatile experimental system to characterize weak and/or transient interactions of membrane-bound proteins proposed to function for OS organization.
We have developed an in vivo protein complementation approach, for use in cultured cells and transgenic X. laevis photoreceptors, that utilizes complementary fragments of yellow fluorescent protein (YFP), to visualize assembly and trafficking of photoreceptor protein complexes. We used this method to investigate protein-protein interactions implicated in the scaffolding of OS architecture in conjunction with a variety of other techniques, including: Western blotting, confocal microscopy, immunoprecipitation, FACS, and electron microscopy.
We observe that YFP-fragment fusion proteins, containing both soluble and membrane-bound glutamic acid rich protein (GARP) variants, can be expressed in HEK293 cells and transgenic X. laevis photoreceptors. When co-expressed with membrane-bound peripherin/rds (P/rds), complementary partner pairs generate fluorescent complexes in both expression systems. Detailed analyses of individual partner and assembled complex distributions suggest that GARP variant assembly with P/rds is correlated with nascent disk morphogenesis.
Our development of an in vivo bimolecular fluorescence complementation approach offers a valuable new strategy for characterizing protein interactions important for photoreceptor OS organization and renewal. In conjunction with other studies, application of this strategy suggests that photoreceptor GARP variants may play at least two roles for OS scaffolding.
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