The Arf•GDP-regulated recruitment of GBF1 to Golgi membranes requires domains HDS1,2 and a Golgi-localized protein receptor

Abstract

We previously proposed a novel mechanism by which the enzyme Golgi-specific BFA resistance factor 1, or GBF1, is recruited to the membranes of the cis-Golgi based on in vivo experiments. Here, we extend the in vivo analysis on production of regulatory Arf•GDP and observe that ArfGAP2/3 do not play such a role. We confirm that Arf•GDP localization is critical as a TGN-localized Arf•GDP mutant protein fails to promote GBF1 recruitment. We also report the establishment of an in vitro GBF1 recruitment assay that supports regulation of GBF1 recruitment by Arf•GDP. This in vitro assay yielded further evidence for the requirement of a Golgi-localized protein since heat denaturation or protease treatment of Golgi membranes abrogated GBF1 recruitment. Finally, combined in vivo and in vitro measurements indicate that the recruitment to Golgi membranes via a putative receptor requires only the HDS1 and HDS2 domains in the C-terminal half of GBF1.