Auto-inhibitory interactions of Sec7, master regulator of the Golgi complex

In eukaryotes, protein and lipid secretory trafficking is orchestrated by the Golgi apparatus where proteins are modified and sorted in a cascade of functionally distinct compartments. The composition of these compartments is maintained primarily by vesicular traffic facilitated by Arf and Rab GTPases. These GTPases are regulated by a GTP/GDP cycle facilitated by Guanine Nucleotide Exchange factors (GEFs) and GTPase Activating Proteins (GAPs) to induce GDP/GTP exchange and GTP hydrolysis respectively. Vesicular trafficking to and from the final compartment, the trans-Golgi network, maintains membrane flux throughout the cell with cargo recycling and secretory pathways. Arf1 and its close paralogs are responsible for virtually all vesicle biogenesis events at the Golgi complex. In budding yeast, Arf1 is activated at the trans-Golgi network by the Arf GEF Sec7, the homolog of the human ARFGEF/BIG proteins. Previous work has determined that Sec7 is regulated by autoinhibition, positive feedback, and GTPase crosstalk, yet the mechanistic basis for these regulatory features is unknown. To understand how Sec7 is regulated, I have determined the structure of the flexible Sec7 dimer by cryoEM at 4.9A resolution. Focused refinements on individual monomer subunits improved resolution to 3.8A, sufficient for molecular model building. This structure reveals the mechanism for Sec7 autoinhibition: in the cytosol, the GEF domain interacts with the HDS domains in a manner that prevents binding to the Arf1 substrate. We also observe that the dimerization region imparts a twist between the two monomers, apparently preventing both catalytic subunits from interacting with the membrane surface simultaneously. Functional studies are currently underway to determine the physiological relevance of our structural findings.