The Golgi Arf-Gef Sec7 Integrates Multiple Signaling Pathways

Abstract

Arf family GTPases are important molecular switches that regulate vesicular trafficking and membrane dynamics in eukaryotic cells. Arf-GDP, the inactive form of Arf, is soluble and only transiently associates with membranes; however, nucleotide exchange catalyzed by an ArfGEF (guanine nucleotide exchange factor) induces a conformational change that allows Arf to tightly interact with membranes through an exposed N-myristoylated [alpha]-helix. Arf-GTP then recruits effectors such as coat proteins and lipid modifying enzymes. The basic mechanisms of GEF activation of Arf are understood, but the mechanisms regulating Arf recruitment to specific membrane compartments have not been investigated in detail. Arf is activated at the Golgi complex by Arf-GEFs of the BIG and GBF families, which are large (180-200 kDa) proteins that share multiple homology domains of unknown function. In Saccharomyces cerevisiae, the BIG/GBF Arf-GEFs include Sec7, Gea1, and Gea2. Sec7 activates Arf1 at the trans-Golgi, but it is largely unknown how Sec7 itself is recruited and regulated. We first determined that the HDS1 domain of Sec7 interacts with Arf1-GTP to stably recruit Sec7 to membranes via positive feedback. I subsequently determined that Sec7 is an effector of two Rab GTPases, Ypt1 (Rab1) and Ypt31/32 (Rab11), which interact with the HDS2-3 region of Sec7. I established that Arf1, Arl1, and Ypt1 primarily affect the membrane localization of Sec7, whereas Ypt31/32 exerts a dramatic stimulatory effect on the nucleotide exchange activity of Sec7. Direct regulation by Arf1, Arl1, and Ypt1 suggests that Sec7 provides a mechanistic link between incoming and outgoing vesicle traffic. Subsequent interactions with Ypt31/32 then further stimulates the activity of Sec7 and drives Sec7-dependent cargo sorting events, providing a mechanistic explanation for Ypt31/32 function in vesicle biogenesis. Taken together, my data suggest that multiple signaling pathways integrate to control both Sec7 recruitment to membranes and Sec7 GEF activity.