May 2006
Volume 47, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2006
The Role of an ARF–GAP, ASAP1, in Rhodopsin Trafficking and Vesicle Budding
Author Affiliations & Notes
  • L.M. Astuto–Gribble
    Dept. of Surgery, Division of Ophthalmology, University of New Mexico, Albuquerque, NM
  • J. Mazelova
    Dept. of Surgery, Division of Ophthalmology, University of New Mexico, Albuquerque, NM
  • N. Ransom
    Dept. of Surgery, Division of Ophthalmology, University of New Mexico, Albuquerque, NM
  • H. Inoue
    Laboratory of Cellular Oncology, National Cancer Institute, Bethesda, MD
  • P. Randazzo
    Laboratory of Cellular Oncology, National Cancer Institute, Bethesda, MD
  • D. Deretic
    Dept. of Surgery, Division of Ophthalmology, University of New Mexico, Albuquerque, NM
  • Footnotes
    Commercial Relationships  L.M. Astuto–Gribble, None; J. Mazelova, None; N. Ransom, None; H. Inoue, None; P. Randazzo, None; D. Deretic, None.
  • Footnotes
    Support  NIH grant EY 12421
Investigative Ophthalmology & Visual Science May 2006, Vol.47, 821. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      L.M. Astuto–Gribble, J. Mazelova, N. Ransom, H. Inoue, P. Randazzo, D. Deretic; The Role of an ARF–GAP, ASAP1, in Rhodopsin Trafficking and Vesicle Budding . Invest. Ophthalmol. Vis. Sci. 2006;47(13):821.

      Download citation file:


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

      ×
  • Supplements
Abstract

Purpose: : The small GTP–binding protein ARF4, a class II ARF, specifically recognizes and binds to the VXPX–COOH sorting motif of rhodopsin. Rhodopsin–ARF4 interaction regulates the budding of rhodopsin transport carriers (RTCs). To test if rhodopsin controls the ARF4–GTPase reaction by regulating the access of an ARF–GAP to ARF4, we sought to identify the ARF–GAP that interacts with ARF4 in retinal photoreceptors.

Methods: : Budding of RTCs was studied using a retinal cell–free system, which reconstitutes rhodopsin trafficking in vitro. ARF1, ARF4, Rab6 and Rab11 were detected by SDS–PAGE and Western blotting with Streptavidin–HRP. Confocal microscopy was performed to determine their subcellular localization.

Results: : By western blotting with specific antibodies, we determined that photoreceptors express high levels of ASAP1, an ARF–GAP that contains ankyrin repeats, a pleckstrin homology (PH) domain, Src homology domain 3 (SH3) and a Bin/amphiphysin/Rvs (BAR) domain that is involved in sensing and/or inducing membrane curvature. ASAP1 has a preference for ARF1, a class I ARF, and ARF4 and ARF5, which are class II ARFs. By confocal microscopy, ASAP1 was localized to punctate structures distributed along the photoreceptor microfilaments and in the vicinity of the Golgi/TGN. Double labeling also demonstrated ASAP1 co–localization with rhodopsin, Rab6 and Rab11 at the Golgi/TGN and on RTCs in the rod inner segment (RIS). A N–terminal fragment of ASAP1 (1–724 aa), containing the BAR domain, PH domain and the ARF–GAP domain, was added to our in vitro retinal cell–free system. The addition of ASAP1 N terminal fragment increased RTC budding by approximately 50% compared to controls.

Conclusions: : Like ARF4, the ARF–GAP ASAP1 regulates budding of RTCs and rhodopsin trafficking in photoreceptor cells.

Keywords: photoreceptors • opsins 
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×