April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
Peripherin-2/rds Function for Outer Segment Structure
Author Affiliations & Notes
  • Andrew F X Goldberg
    Eye Research Institute, Oakland University, Rochester, MI
    Department of Biological Sciences, Oakland University, Rochester, MI
  • Melanie N Gary
    Eye Research Institute, Oakland University, Rochester, MI
  • Mark E English
    Eye Research Institute, Oakland University, Rochester, MI
  • Linda Ritter
    Eye Research Institute, Oakland University, Rochester, MI
  • Footnotes
    Commercial Relationships Andrew Goldberg, None; Melanie Gary, None; Mark English, None; Linda Ritter, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 419. doi:
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      Andrew F X Goldberg, Melanie N Gary, Mark E English, Linda Ritter; Peripherin-2/rds Function for Outer Segment Structure. Invest. Ophthalmol. Vis. Sci. 2014;55(13):419.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Vertebrate vision relies on photon capture by the flattened membranous disks that comprise rod and cone outer segments (OSs). The mechanisms that generate and maintain the membrane curvature required for normal disk morphology are not yet defined, although the photoreceptor-specific integral membrane protein peripherin/rds (P/rds) is thought to play a role. The aim of this study was to investigate the recent proposal that an induced amphipathic helix (AH) within the cytoplasmic C-terminal domain of P/rds can contribute to the membrane curvature of OS disks.

Methods: Association of the P/rds C-terminal domain (CTER) with membranes was assayed in vitro using extruded liposomes in conjunction with purified recombinant proteins. A gradient-based floatation assay was used to characterize protein-membrane interaction and derive partition coefficients. The contribution of this domain to membrane curvature generation was assessed in cellulo, using WT and mutant variants of P/rds expressed in cultured cells, in conjunction with immunofluorescence analyses using laser scanning confocal microscopy and ultrastructural analyses using electron microscopy.

Results: CTER associated avidly with liposomes generated from lipids extracted directly from purified OS membranes. It likewise showed a strong association with liposomes generated from a mixture of synthetic phospholipids based on OS disk membranes. In contrast, a significantly weaker association was observed for liposomes generated from a brain polar lipid extract. Lipid substitution and protein mutagenesis studies found that avid association required membrane surface defects (conically-shaped lipids) and was promoted by (anionic) surface charge. Full-length P/rds and a deletion mutant lacking only the inducible AH, were each released from the cellular secretory pathway when expressed in cultured cells, and accumulated within networks of high-curvature membrane tubules.

Conclusions: We found lipid composition to be a key regulator of P/rds cytoplasmic C-terminus interaction with membranes. In particular, the results indicate an essential role for conically-shaped phospholipids (such as DHA-containing species), which introduce surface packing defects into membranes. These findings are consistent with a hydrophobic insertion (wedging) model for curvature generation, and support a model in which the C-terminal AH contributes to disk rim formation/stability in parallel with other protein domains.

Keywords: 648 photoreceptors • 659 protein structure/function • 695 retinal degenerations: cell biology  
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