Abstract
The Golgi complex is the central membrane and protein-sorting station in eukaryotic cells. Activation of Arf (ADP-ribosylation factor) GTPases is essential for vesicle formation via recruitment of cargo adaptors and coat proteins necessary for Golgi trafficking. Arf activation is spatially and temporally regulated by distinct guanine nucleotide exchange factors (GEFs) at different Golgi compartments. The yeast Arf-GEF Sec7 is a conserved and essential activator of Arf1 at the trans-Golgi network. Sec7 contains a highly conserved regulatory region, the homology upstream of Sec7 (HUS) box, with an unknown mechanistic role. In this study we explore how the HUS box, which is N-terminal to the catalytic domain, acts together with C-terminal regulatory domains in the allosteric activation of Sec7. We report that mutation of the HUS box disrupts positive feedback and allosteric activation of Sec7 by the GTPase Ypt31, a yeast Rab11 homolog. Taken together, our results support a model in which the inter- and intramolecular interactions of the HUS box and the C terminus are necessary for the allosteric activation of Sec7.