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Michelle Lynn Milstein, Victoria A. Kimler, Chiranjib Ghatak, Alexey S. Ladokhin, Andrew F X Goldberg; Molecular determinants of peripherin-2/rds membrane-shaping activity. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1029.
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© ARVO (1962-2015); The Authors (2016-present)
We are investigating the molecular mechanisms by which peripherin-2/rds (P/rds) supports rod and cone photoreceptor outer segment (OS) structure, and how inherited defects in this protein can lead to a variety of progressive retinal degenerations. Our previous studies suggest that P/rds may help to shape the high curvature rim domains of OS disks. Here, we investigate the structural determinants that contribute to this activity.
Biochemical, biophysical, and imaging techniques were used to investigate the role of the P/rds C-terminal domain and a proposed inducible amphipathic helix (AH) contained within it. Key Recombinant constructs were P/rds△AH, a deletion mutant lacking the residues encoding the proposed inducible AH, and CTER, a soluble ~7 kDa recombinant protein corresponding to the cytoplasmic P/rds C-terminus. Protein biosynthesis, trafficking, subcellular localization, and impact on membrane ultrastructure were analyzed by velocity sedimentation, ICC/IHC, and TEM respectively.
Loss of the inducible C-terminal AH did not impede P/rds△AH biosynthesis, tetrameric polymerization, post-translational processing, or GARP2 binding in HEK AD293 cells. AH deletion did not adversely affect trafficking or localization of P/rds△AH in transgenic vertebrate photoreceptors; P/rds△AH localized solely to OSs with disk rim localization, in a manner largely independent of the endogenous WT P/rds. Finally, although we found that bona fide phospholipid membranes can induce a coupled folding/partitioning of an AH in the P/rds C-terminus, this motif was not required for the generation of membrane curvature in cellulo.
This study demonstrates that a membrane-inducible AH in the P/rds C-terminus is not required for protein biosynthesis, subunit assembly, higher-order polymerization, targeting to and localization at OS disk rims, or the generation of membrane curvature. Our results further suggest that C-terminal regions outside of the AH motif mediate P/rds targeting and protein-protein interactions, and that deletion of this motif may activate P/rds curvature generating activity, suggesting a potential regulatory role for this motif in OSs. Together with previous studies, our results suggest that inherited defects affecting the P/rds C-terminus can act via one or more of several mechanisms (e.g. altering GARP2 binding, dysregulating curvature generating activity, and/or impairing protein targeting).
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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