Abstract
Purpose :
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.
Methods :
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.
Results :
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.
Conclusions :
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.