Field of Biochemistry, Molecular and Cell Biology Ph.D. Thesis Defense Seminar

Bryce Brownfield

Fromme Lab

Wednesday, May 31, 2023, 10:00 AM

146 Stocking Hall

Zoom Meeting Link:

https://cornell.zoom.us/i/987371103602pwd=bFpiZ2J1Yv80dWZROEpRROFXSEcxZz.09

Meeting ID: 987 3711 0360

Passcode: 418927

6A Tale of Two Conformations: How the Arf GEF Sec7 Manages

Autoinhibition”

The Golgi apparatus is a highly modular organelle that mediates the secretory pathway in eukaryotic cells The final compartment, the trans-Golg1 Network (IGN). connects the Golgi to the endomembrane svstem and balances the flow of membranes and cargo throughout the cell. Virtually all sorting and transport at the G\ is denendent on the smal Pase Art which omves vesice tormation and membrane moditication following its activation b the Art GEF Sec7. A network of inter- and intramolecular interactions of Sec7 gives robust spatiotemporal specificity to the potent Arf GEF. The SecT homodimer is autoinhibited, and interactions with Rabl, Rabil, Arll, Artl and the membrane recruit Sec7 and stimulate activity. These interactions have been mapped biochemicall to the regulatory domains of Sec7. but the precise mechanism of autoregulation has not been established. To identify the intramolecular interactions regulating Sec7, we determined the structure of full length Sec7 to 3.7 A by cro electron microscopy. This structure reveals an interaction between the GEF and HDS2 domains occludes the catalvtic surface, and disruptions to this interface results in higher GEF activity in vitro and growth defects in vivo. Serendipitously, the AlphaFold predicted structure naced the Che domain actacent to the CB/HIS domain and we demonstrate this is the active conformation. In this conformation the GEF domain displaces a loop in the DCB/HUS domain we refer to as the “D-loop” Removal of the D-loop results in a modest increase in GEF activity in vitro. and when combined with HDS2 mutations synergistically increases activity and disrupts physiological function coincident With the severitv of the HDsz distuption. lhe structure also reveals a hydrophobic surtace in the

His< domain is the dimerzation intertace.

and we find dimerization and switching to the active

conformation modulates membrane avidity. An amphipathic Helix in the HDS1-2 linker essential for Gea2 localization contributes but is not recured for the sec membrane interaction. we therefore conclude the GEE domain is in eautomum between active and automhibited contormations governed ow the attnitw ot

the GEr-HIS? interaction the -loon and the membrane