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Qin Liu, Eric A. Pierce; Identification Of Rp1 Interacting Proteins Using In Vivo Affinity Purification Approaches. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1633.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations in the RP1 gene cause both dominant and recessive RP. So far, the function of RP1 protein and the mechanism by which mutations in RP1 lead to photoreceptor death remain elusive. In attempting to fully understand the function of the Rp1 protein, we generated lines of transgenic mice that express Rp1 proteins with N- and C- affinity purification epitopes. We then used affinity purification methods to identify proteins that interact with Rp1 in vivo.
Using a BAC clone containing mouse Rp1 genomic DNA, we fused a SF-TAP (Strep-tag II-FLAG) tag to the N- or C-terminus of the Rp1 coding sequence to generate transgenic mice that express epitope-tagged versions of the full-length Rp1 protein, N-TAP-Rp1 and C-TAP-Rp1. Retinas from both transgenic mice and non-transgenic controls were extracted and affinity purified using anti-flag antibody. The purified protein complexes were subjected to mass spectrometry analyses and the components of the complex from both lines were compared to generate the list of candidate Rp1 interacting proteins.
Our data indicate that the N-TAP-Rp1 and C-TAP-Rp1 proteins localize correctly to the axonemes of photoreceptor cells and function normally. A total of 14 proteins were detected in both N-TAP-Rp1 and C-TAP-Rp1 retinas, but not in the wild type controls. Of those, Rp1l1 was the most abundant protein detected in the complex. Of particular interest, we found several chaperone and co-chaperone proteins in the Rp1 protein complex, including three members of Hsp70 family and two bcl-2-associated athanogene (BAG) proteins.
We have identified several interacting proteins of Rp1 by TAP approaches. Rp1l1 was previously reported to co-localize and interact with Rp1. However, function of Rp1l1 protein in photoreceptor cells is not understood. Chaperone proteins are reported to be widely distributed in the cilia and flagella, and are potentially related to axonemal protein dynamics. Further evaluation of the significance of the interactions between Rp1 and the candidate interacting proteins identified to date is in progress.
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