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D Deretic, V Morel; Proteins Interacting with the C-terminal Targeting Signal of Rhodopsin . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1386.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Mutations clustered at the C-terminal of rhodopsin appear to cause severe forms of autosomal dominant retinitis pigmentosa (ADRP). The C-terminal sequence of rhodopsin QVS(A)PA is recognized by specific factor(s) in the trans-Golgi network (TGN) and regulates sorting and post-Golgi trafficking of rhodopsin reconstituted in the frog retinal cell-free system (RCFS) (Deretic et al., PNAS 95, 10620, 1998). In the present study we searched for the protein(s) that recognize and bind to the rhodopsin C-terminal sorting signal. Methods: Using the established RCFS we performed cross-linking experiments and GST pull down assays. Proteins interacting with the C-terminal sequence were also detected by Far Western overlay. Results: A cross-linked product of ∼50 kDa containing rhodopsin could be obtained in a time dependant fashion using a cleavable photoactivatable membrane-impermeable crosslinker sulfo-SADP in the RCFS. The cross-linker cleavage by beta-Mercaptoethanol revealed the presence of a doublet of proteins around 25 kDa and a less abundant protein of 21 kDa. The time course of the crosslinking was consistent with the binding of these proteins to rhodopsin cytoplasmic C-terminal during post-Golgi membrane budding from the TGN. The 50 kDa cross-linked product could still be obtained after budding, in the fraction enriched in post-Golgi membrane carriers. Interacting candidates in the 21 kDa range were detected by GST pull down assay with the GST fusion protein containing the full C-terminal tail of rhodopsin (GST-Rh-C) but not with the one in which the last five amino acids were truncated (GST-Rh-CT). Another interacting candidate was detected after Far Western overlay of retinal PNS using the same full length GST protein (GST-Rh-C). We are analyzing the protein composition of these complexes. Conclusion: Our data suggest that macromolecular complexes that regulate intracellular trafficking may bind to rhodopsin. Some components of these protein complexes may specifically recognize the last five amino acids of rhodopsin and regulate its entry into post-Golgi carriers as they bud from the TGN.
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