Abstract
Purpose :
Photoreceptor outer segment (OS) disk rims are characterized by an extreme membrane curvature that is generated and stabilized by a transmembrane scaffold comprising three parallel rows of dimers containing peripherin-2 and/or rom1. The protein-protein interactions underlying scaffold organization and function are not well understood but are essential for retinal health and normal vision. The present studies are aimed at addressing this knowledge gap.
Methods :
Purified rod outer segments (ROS) were lysed in 1% Triton X-100 or 1% DDM, and soluble extracts were collected by centrifugation. We redesigned a velocity sedimentation method, originally established to assay peripherin-2 stoichiometry, to expand its dynamic range and improve reproducibility. A native PAGE method was adapted to resolve the size species present in sedimentation fractions, in unfractionated ROS, and in soluble extracts of transfected HEK293 cells. Recombinant peripherin-2 mutants were used as calibration standards for assigning size-species via native PAGE.
Results :
We applied the redesigned sedimentation assay to soluble extracts from bovine and murine ROS membranes. In each case, peripherin-2 sedimented in a heavy and a light peak, and rom1 sedimented only in the light peak. This was true whether membranes were solubilized with Triton X-100 or DDM. We applied native PAGE to resolve the size species present in sedimentation fractions and found surprising heterogeneity within each peak. Unfractionated soluble ROS extracts produced a laddering pattern consistent with that of the fractionated samples. In each case, peripherin-2 polymers ranged from dimers to eicosamers and rom1 polymers ranged from dimers to hexamers. Chemical reduction of solubilized ROS extracts prior to native PAGE collapsed higher order polymers, leaving peripherin-2 tetramers and rom1 dimers as the predominant species. Similarly, ladders produced by recombinant peripherin-2 could be collapsed by a C150S mutation.
Conclusions :
Peripherin-2 and rom1 polymers solubilized from OS disk rim scaffolds or transfected HEK 293 cells are more heterogeneous than previously understood. Results support our previous model of disulfide-linked polymer chains and reveal previously undocumented non-covalent interactions. This new combination of sedimentation and native PAGE will be useful for analyzing the impact of pathogenic PRPH2 and ROM1 mutations.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.