June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
SNAREs interact with RDS/ROM-1 during outer segment targeting
Author Affiliations & Notes
  • Rahel Zulliger
    Cell Biology, OUHSC, Oklahoma City, OK
  • Shannon Conley
    Cell Biology, OUHSC, Oklahoma City, OK
  • Maggie Yoder
    Cell Biology, OUHSC, Oklahoma City, OK
  • Seifollah Azadi
    Ophthalmology, OUHSC, Oklahoma City, OK
  • Muna I Naash
    Cell Biology, OUHSC, Oklahoma City, OK
  • Footnotes
    Commercial Relationships Rahel Zulliger, None; Shannon Conley, None; Maggie Yoder, None; Seifollah Azadi, None; Muna Naash, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 5502. doi:
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    • Get Citation

      Rahel Zulliger, Shannon Conley, Maggie Yoder, Seifollah Azadi, Muna I Naash; SNAREs interact with RDS/ROM-1 during outer segment targeting. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5502.

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

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Abstract

Purpose: Peripherin-2, also known as RDS (retinal degeneration slow), is an important structural protein in the photoreceptor outer segment (OS), and mutations in it cause several severe forms of retinal degeneration. Together with its non-glycosylated homolog ROM-1 (rod outer segment protein 1), RDS is synthesized in the inner segment and then trafficked into the outer segment where it functions in non-covalently linked tetrameric complexes as well as covalently linked larger complexes. However, in spite of the fact that many RDS mutations interrupt OS targeting, little is known about the processes which govern RDS OS targeting.

Methods: To investigate possible trafficking partners of RDS and ROM-1, we analyzed the bound fraction of RDS and ROM-1 pulldown by mass spectrometry. Potential candidates were confirmed with in vivo and in vitro binding assays. Immunohistochemistry (IHC) was performed to show co-localization in the retina and proximity ligation assay (PLA) was conducted to show interactions in situ.

Results: We used biochemical and cell biological techniques to show that RDS/ROM-1 interact with two proteins known to be involved in OS targeting of other proteins (such as rhodopsin), namely Syntaxin 3B and SNAP-25 from the SNARE family. The interaction was initially seen with mass spectrometry and was confirmed by co-immunoprecipitation in in vivo and in vitro. The IHC showed a possible site of interaction at the apical edge of the inner segment and binding between Syn3B and RDS in this region was confirmed in situ using the PLA. We further show that Syn3B interacts with both covalently linked and non-covalently linked RDS complexes.

Conclusions: Our data suggest that the RDS/ROM-1 complexes are trafficked in vesicles that utilize Syn3B and SNAP-25 for fusion with the inner segment membrane during OS targeting. Furthermore, the interaction of Syn3B with covalently linked RDS complexes indicates that RDS/ROM-1 complex assembly occurs prior to arrival of these proteins in the OS, suggesting that the ability to form normal complexes may be a requirement for proper trafficking. This OS trafficking process also involves other proteins which will be the focus of future research.

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