Abstract
Purpose:
The small GTPase Arf4 and the Arf GAP ASAP1 cooperatively regulate budding of rhodopsin transport carriers (RTCs) from the Golgi/TGN during ciliary trafficking. In this study we wanted to determine if in these processes Arf4 is activated by the Arf guanine nucleotide exchange factor (GEF) GBF1, and examine the role of rhodopsin in the activation of Arf4.
Methods:
The distribution of GBF1 and its interactions with rhodopsin and Arf4 were examined by pulse-chase experiments, retinal subcellular fractionation, confocal microscopy and the Proximity Ligation Assay (PLA), which detects protein-protein interactions in situ.
Results:
The Arf GEF GBF1 mainly co-localized with the Rab6 GTPase at the photoreceptor trans-Golgi. The PLA analysis revealed that Arf4 and, surprisingly, rhodopsin interact with GBF1, nearly exclusively at the Golgi/TGN. Rhodopsin-GBF1, Arf4-GBF1 and rhodopsin-Arf4 interactions were all significantly disrupted by the treatment of retinas with Golgicide A (GCA), a selective inhibitor of GBF1, indicating that GBF1 is essential for the activation of Arf4. Similar to the BFA inhibition of all Arf GEFs, the GCA inhibition of GBF1 caused accumulation of rhodopsin in the Golgi and significantly slowed down trafficking to the cilium and the ROS. However, unlike in BFA-treated retinas where Golgi was disassembled, in GCA-treated retinas it retained its regular appearance, indicating that GBF1 is not involved in the maintenance of the photoreceptor Golgi. Surprisingly, Arf GEF GBF1 directly interacted with the Arf GAP ASAP1 and the interaction sites were detected both at the Golgi/TGN and on RTCs, despite the lack of Arfs on RTCs. Moreover, the GEF activity of GBF1 was not important for the GBF1-ASAP1 interface, as GCA had no effect on their interaction.
Conclusions:
The Arf GEF GBF1, Arf4 and the Arf GAP ASAP1 form a functional network that regulates the Golgi export of rhodopsin and potentially other ciliary cargo. The influx of rhodopsin into the trans-Golgi cisternae is a signal that GBF1 recognizes to activate Arf4 during membrane trafficking to primary cilia.
Keywords: 648 photoreceptors •
659 protein structure/function •
660 proteins encoded by disease genes