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Dimitra Athanasiou, Maria Kosmaoglou, Naheed Kanuga, Sergey Novoselov, Adrienne W. Paton, James C. Paton, Paul Chapple, Michael E. Cheetham; The Molecular Chaperone BiP Maintains Rod Opsin Mobility in the Endoplasmic Reticulum. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1584.
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© ARVO (1962-2015); The Authors (2016-present)
Molecular chaperones, the facilitators of proper protein folding are important for photoreceptor function. Overexpression of BiP can alleviate the toxic effects of misfolded rod opsin in the retina (Gorbatyuk et al., 2010). In this study, we investigated the role of BiP on wild-type (WT) and mutant (P23H) rod opsin biogenesis by manipulating the BiP levels in a cell culture model of rod opsin processing.
The localisation of WT and P23H rod opsin was investigated in the presence of the subtilase cytotoxin SubAB or with overexpression of WT BiP and mutant (T37G) BiP in SK-N-SH neurobastoma cells. Co-immunoprecipitation studies were performed on either transfected cell lysates or porcine retina lysates. Rod opsin ubiquitylation status was monitored by co-localisation and co-immunoprecipitation. Rod opsin mobility in the ER was investigated using fluorescence recovery after photobleaching (FRAP).
BiP levels were specifically depleted to less than 10% of control by treatment with the subtilase cytotoxin SubAB. BiP depletion resulted in ER retention of WT rod opsin and reduced traffic to the plasma membrane. Co-immunoprecipitation studies in cells and retinal lysates revealed a weak but specific interaction between BiP and WT rod opsin. SubAB mediated ER retention of WT rod opsin led to an increase in rod opsin ubiquitylation. Investigation of rod opsin mobility by FRAP revealed that treatment with SubAB, or co-expression of a BiP ATPase mutant, BiP(T37G), compromised the WT rod opsin mobility. Interestingly, WT rod opsin mobility was reduced to below the mobility of the misfolded P23H rod opsin, which is retained in the ER and forms ubiquitylated inclusions in the cytoplasm. SubAB treatment of cells expressing P23H rod opsin decreased the mobility of the mutant protein further and led to ubiquitylation throughout the ER. Interestingly, overexpression of WT but not T37G BiP improved the mobility of P23H-GFP in the ER.
BiP is important for rod opsin biogenesis. Inhibition of BiP function results in aggregation of WT rod opsin in the ER and suggests that BiP might be important for maintaining the solubility of both WT and mutant rod opsin in the ER. Furthermore, BiP overexpression might be used to reduce P23H rod opsin aggregation.
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