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NOVEL REGULATORY MECHANISM OF THE ARF-GEF SEC7 VIA THE N- AND C- TERMINUS

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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 is composed of seven domains. The N-terminal domains, Dimerization and Cyclophilin Binding and Homology Upstream of Sec7 Domains (DCB-HUS), have been implicated in homodimerization, recruitment to the Golgi, and stimulation of GEF activity. The C-terminal domains, Homology Downstream of Sec7 Domain (HDS1-4), are necessary for autoinhibition, recruitment to the Golgi, and allosteric activation. However, the function of a highly conserved N-terminal region referred to as the HUS-box has not been determined. In this work, I report that mutation of the HUS box compromises Sec7 activity on membranes in vitro and is lethal in vivo. I discovered that the cause of this phenotype is disruption of the positive feedback and allosteric activation of Sec7 by the GTPase Ypt31, a yeast Rab11 homolog. These 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. I demonstrate that the established role of the N-terminal region in dimerization is not conserved; instead, a C-terminal autoinhibitory domain is responsible for dimerization of Sec7. Based on unpublished data, I propose that additional regions of Sec7 are responsible for its regulation and suggest future experiments to help decipher additional regulatory mechanisms of Sec7.

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2018-08-30

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Keywords

Cellular biology; allosteric regulation; GTPase; Golgi; Biochemistry; ADP ribosylation factor (ARF); Guanine nucleotide exchange factor (GEF); intracellular trafficking

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Fromme, Joseph Chris

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Brown, William J.
Mao, Yuxin

Degree Discipline

Biochemistry, Molecular and Cell Biology

Degree Name

Ph. D., Biochemistry, Molecular and Cell Biology

Degree Level

Doctor of Philosophy

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Government Document

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Attribution-NonCommercial-NoDerivatives 4.0 International

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dissertation or thesis

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