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D. Deretic, J. Mazelova, L. Astuto-Gribble; The Binding of Rhodopsin C-terminus to ARF4 Regulates the Assembly of a Molecular Complex That Includes the ARF-GAP ASAP1 and the GTPase Rab11. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4669.
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© ARVO (1962-2015); The Authors (2016-present)
The incorporation of rhodopsin into specialized transport carriers (RTCs) is regulated by the direct binding of its C-terminal sorting signal, VXPX-COOH, to the small GTP-binding protein ARF4. One of the downstream effectors that regulates RTC budding from the Golgi/TGN by increasing membrane curvature is an ARF-GAP ASAP1. We find that ASAP1 co-distributes and co-fractionates with the small GTPase rab11, previously localized to RTCs. In this study we further examined the ARF4-ASAP1-rab11 interaction in rhodopsin trafficking.
RTC budding was evaluated using an established cell-free assay. Subcellular localization of ASAP1 and rab11 was determined by confocal microscopy.
The inhibition of RTC budding in the cell-free system with an anti-ARF4 blocking antibody resulted in the accumulation of ARF4 on the Golgi/TGN membranes accompanied by the release of rab11 into the cytosol, suggesting an involvement of ARF4 in the recruitment of rab11 to RTC budding sites. RTC budding in the cell-free assay was decreased by 30% with three-fold reduction of rab11 by cytosol immunodepletion. By confocal microscopy, rab11 was localized to punctate structures distributed along the Golgi/TGN, consistent with RTC budding sites, where it co-localized with ASAP1 and Arfophilin1/FIP3, an ARF/rab11 binding protein. Thus, ARF4 may recruit ASAP1 and rab11, through FIP3, to RTC budding sites. Subcellular fractionation and co-immunoprecipitation experiments showed sequential membrane dissociation of ARF4, followed by ASAP1 and FIP3, and the retention of rab11 on RTCs.
Our data suggest that ASAP1, FIP3 and rab11 may be a part of a macromolecular complex whose assembly is regulated by the binding of ARF4 to rhodopsin. This complex may couple rhodopsin sorting, formation of RTCs, and concomitant recruitment of the molecular machinery necessary for RTC fusion. The presence of rab11 on RTCs may subsequently ensure the recruitment of the Sec6/8 tethering complex involved in RTC fusion, and/or the recruitment of molecular motors such as myosin V, known to interact with rab11.
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