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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.
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© ARVO (1962-2015); The Authors (2016-present)
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.
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.
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.
Like ARF4, the ARF–GAP ASAP1 regulates budding of RTCs and rhodopsin trafficking in photoreceptor cells.
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