June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Outer Segment Targeting of R9AP in Mouse Rod Photoreceptors
Author Affiliations & Notes
  • Jillian Pearring
    Duke Eye Center, AERI, Duke University, Durham, NC
  • Sidney Gospe
    Department of Pharmacology and Cancer Biology, Duke Univsersity, Durham, NC
  • Sheila Baker
    Department of Biochemistry, University of Iowa, Iowa City, IA
  • Vadim Arshavsky
    Duke Eye Center, AERI, Duke University, Durham, NC
    Department of Pharmacology and Cancer Biology, Duke Univsersity, Durham, NC
  • Footnotes
    Commercial Relationships Jillian Pearring, None; Sidney Gospe, None; Sheila Baker, None; Vadim Arshavsky, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 2644. doi:
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      Jillian Pearring, Sidney Gospe, Sheila Baker, Vadim Arshavsky; Outer Segment Targeting of R9AP in Mouse Rod Photoreceptors. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2644.

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

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Abstract

Purpose: Our laboratory is engaged in identification of the targeting signals within the outer segment-specific proteins that ensure their proper subcellular localization. We previously showed that in Xenopus rods the outer segment serves as the default trafficking destination for membrane proteins which do not contain specific targeting signals, and that this default delivery pattern appears to be sufficient to explain the intracellular localization of the protein R9AP. However, in mouse rods, the outer segment does not serve as a dominant destination for untargeted membrane proteins, which led us to re-visit the targeting of R9AP in these cells.

Methods: GFP and FLAG-tagged mouse R9AP, R7BP and their chimeric proteins were cloned into the pRho vector, containing the 2.2 kb bovine rhodopsin promoter to drive expression in mouse rods. Plasmid DNA was electroporated into photoreceptors of neonatal mice and intracellular distribution of the expressed protein constructs was analyzed at P21 by confocal microscopy. Transgenic Xenopus constructs utilized a dual promoter strategy; Xenopus opsin promoter was used to express FLAG or myc-tagged constructs in rods, while a γ-crystallin promoter was used to drive GFP expression in the lens in order to identify positive tadpoles. Subcellular localization of the expressed constructs was analyzed at developmental stage 45 by confocal microscopy.

Results: By analyzing FLAG-tagged R9AP constructs expressed in mouse rods, we identified two putative regions on R9AP involved in its outer segment delivery. Constructs representing R9AP fragments containing either the tri-helical bundle or the SNARE homology domain (attached to the membrane via a single-pass transmembrane domain or a lipid anchor) resulted in normal outer segment localization. However, only the tri-helical bundle of R9AP was able to confer outer segment localization of a chimeric construct between R9AP and a homologous protein, R7BP. The latter was also demonstrated to take place in Xenopus rods.

Conclusions: Our findings indicate that R9AP is not reliant on default protein trafficking flux for its outer segment delivery in mammalian and perhaps also frog rods, but contains its own targeting signal likely shared between the tri-helical bundle and the SNARE homology domain.

Keywords: 648 photoreceptors • 659 protein structure/function  
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