Assembly of cytosolic coat proteins I (COPI) complex at the ER-Golgi interface is directed by the small GTPase ADP ribosylation factor 1 (Arf1) and its guanine nucleotide exchange factor, GBF1. Our previous results indicated that overexpression of the GTP-restricted Rab1 mutant (Rab1Q67L) increases association of GBF1 and COPI in ER exit sites interface. Moreover, an interaction between Rab1 and GBF1 was reported. Here, we further characterized this interaction. The ability of recombinant Rab1 to bind either the N-terminal or the C-terminal domains of GBF1 was tested using GST-pull down assays. We show that GTP form of Rab1 directly binds the N-terminal domain of GBF1. To evaluate how Rab1-GBF1 interaction influences Arf1, we measured Arf1 dynamics in presence of active Rab1. We performed FRAP assays in cells co-expressing Arf1-GFP and Rab1Q67L. Arf1 membrane association-dissociation kinetics at the Golgi complex was delayed. Moreover, Rab1Q67L inhibits Arf1 membrane dissociation induced by Brefeldin A suggesting that Rab1 modulates Arf1 membrane association. Taken together our data support a model where Rab1 directly interacts with the N-terminal domain of GBF1 to modulate Arf1 dynamics and activity.
