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Andrew F X Goldberg, Michelle Lynn Milstein, Breyanna Lynn Cavanaugh, Stefanie Volland, David S Williams; Peripherin-2/rds functions for photoreceptor disk morphology by bending membranes. Invest. Ophthalmol. Vis. Sci. 2019;60(9):573.
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© ARVO (1962-2015); The Authors (2016-present)
The distinctive architecture of photoreceptor outer segment (OS) organelles is characterized by a regular stacking of many hundreds of pita bread-shaped membranous disks. OS biogenesis and structure require peripherin-2/rds (P/rds), an integral membrane protein that resides at the highly curved rim domain that defines disk peripheries. The molecular function and mechanism of P/rds support for OS structure remain undefined; this protein has been proposed to act as a fusion protein, as an adhesion molecule, as a molecular zipper, and as a generator of membrane curvature. This study was designed to determine the primary molecular function of P/rds for mature OS disks.
We applied a variety of approaches to investigate P/rds, including native protein purification, heterologous expression in HEK293 cells, transmission electron microscopy (TEM), TEM-tomography, image analysis, and reconstitution. We tested the ability of P/rds to generate membrane curvature under a variety of in vitro and in cellulo conditions, and correlated our findings with molecular genetic data and protein structure, to generate a novel model for P/rds shaping of OS disk membranes.
We confirmed that deletion of an inducible C-terminal amphipathic helix dramatically upregulates P/rds activity for generating membrane curvature in cellulo. Activated P/rds generated large 3D networks of membranous tubules, and TEM-tomography of transfected HEK293 cells demonstrated that tubule geometry was directly correlated with P/rds self-assembly state. Normal assembly of P/rds produced extended membrane tubules with diameters similar to those of OS disk rims; however, stage-specific blockade of P/rds self-assembly resulted in zero-length small-diameter tubules, or no small-diameter membranes at all. Together with imaging of protein purified from native OS membranes, the new findings reveal how P/rds activity and organization produce highly curved membranes at disk rims.
This study provides clear evidence that P/rds is a potent generator of membrane curvature, that this activity is driven by multiple stages of protein self-assembly, and that it shapes the highly-curved rims of mature OS disks. In addition to elucidating the primary molecular function of P/rds, the current findings provide the first mechanistic explanation of how mutations in P/rds can cause ultrastructural defects in photoreceptor OS organelles.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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