Regulatory domains of the Arf GEF Sec7 scaffold autoinhibited and active conformations

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The Golgi apparatus is a highly modular organelle that mediates the secretory pathway in eukaryotic cells. The final compartment, the trans-Golgi Network (TGN), connects the Golgi to the endomembrane system and balances the flow of membranes and cargo throughout the cell. Virtually all sorting and transport at the TGN is dependent on the small GTPase Arf1 which drives vesicle formation and membrane modification following its activation by the Arf GEF Sec7. A network of inter- and intramolecular interactions of Sec7 gives robust spatiotemporal specificity to the potent Arf GEF. The Sec7 homodimer is autoinhibited, and interactions with Rab1, Rab11, Arl1, Arf1 and the membrane recruit Sec7 and stimulate activity. These interactions have been mapped biochemically to the regulatory domains of Sec7, but the precise mechanism of autoregulation has not been established. To identify the intramolecular interactions regulating Sec7, I determined the structure of full length Sec7 to 3.7 Å by cryo electron microscopy. This structure reveals an interaction between the GEF and HDS2 domains occludes the catalytic surface, and validate this interaction in vitro and in vivo. Serendipitously, the AlphaFold predicted structure placed the GEF domain adjacent to the DCB/HUS 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 severity of the HDS2 disruption. The structure also reveals a hydrophobic surface in the HDS4 domain is the dimerization interface. We show dimerization, an amphipathic helix in the HDS1-2 linker, and switching to the active conformation modulates membrane avidity. We therefore conclude the GEF domain is in equilibrium between active and autoinhibited conformations governed by the affinity of the GEF-HDS2 interaction, the D-loop, and the membrane.

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210 pages


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Union Local


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Fromme, Joseph

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Hollopeter, Gunther
Kawate, Toshimitsu

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Biochemistry, Molecular and Cell Biology

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Ph. D., Biochemistry, Molecular and Cell Biology

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Doctor of Philosophy

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dissertation or thesis

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