June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
In-coincidence detection of the Golgi influx of rhodopsin and Arf4-GTP by the Arf GEF GBF1 regulates activation of Arf4 in trafficking to cilia and ROS
Author Affiliations & Notes
  • Dusanka Deretic
    Surgery, Univ of New Mexico Sch of Med, Albuquerque, NM
  • Jing Wang
    Surgery, Univ of New Mexico Sch of Med, Albuquerque, NM
  • Footnotes
    Commercial Relationships Dusanka Deretic, None; Jing Wang, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 5526. doi:
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      Dusanka Deretic, Jing Wang; In-coincidence detection of the Golgi influx of rhodopsin and Arf4-GTP by the Arf GEF GBF1 regulates activation of Arf4 in trafficking to cilia and ROS. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5526.

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

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Abstract

Purpose: The small GTPase Arf4 regulates budding of rhodopsin transport carriers (RTCs) from the Golgi/TGN during ciliary trafficking. In this study we wanted to determine the role of rhodopsin and the Arf guanine nucleotide exchange factor (GEF) GBF1 in the activation of Arf4 in this process.

Methods: The distribution of GBF1 and its interactions with rhodopsin and Arf4 were examined by pulse-chase experiments, retinal subcellular fractionation, pulldown assays, confocal microscopy and the Proximity Ligation Assay (PLA).

Results: Arf4 and rhodopsin interacted with GBF1 nearly exclusively at the Golgi/TGN. Surprisingly, Arf GEF GBF1 directly interacted with the Arf GAP ASAP1 both at the Golgi/TGN and on RTCs, despite the lack of Arfs on RTCs. The GBF1 GEF activity was essential for the GBF1-rhodopsin-Arf4 interaction, since its selective inhibitor Golgicide A (GCA) caused a significant decrease in all but the GBF1-ASAP1 interactions. GCA inhibition of GBF1 caused accumulation of rhodopsin in the Golgi and significantly slowed down trafficking to the cilium and the ROS. The GBF1 GEF activity involves the catalytic Sec7 domain. GBF1 also contains conserved regulatory domains: a dimerization and cyclophillin binding (DCB), a homology upstream of Sec7 (HUS), and three homology downstream of Sec7 (HDS) domains. GST-pulldowns of purified rhodopsin were performed with DCB-HUS and Sec7-HDS1, with or without recombinant Arf4 pre-loaded with GTPγS or GDPβS. GST-DCB-HUS pulled down rhodopsin significantly better than GST-Sec7-HDS1, or GST alone, either in the presence or absence of Arf4, demonstrating a direct rhodopsin-GBF1 interaction. Arf4·GTP preferentially interacted with DCB-HUS, whereas Arf4·GDP interacted with Sec7-HDS1. Thus, GBF1 combines a regulatory binding site within its DCB-HUS domain with a catalytic binding site within the Sec7 domain and acts both as a GEF and an effector of Arf4.

Conclusions: Our findings suggest that GBF1 integrates the input from both the influx of rhodopsin into the Golgi/TGN and the activated Arf4, to create a pool of activated Arf4 surrounding the nascent buds that generate ciliary-targeted RTCs. Through a positive feedback loop, GBF1 both amplifies the spatially restricted activation of Arf4 and, with an input from ASAP1, coordinates Golgi export of rhodopsin that is specifically targeted to primary cilia.

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