All ETDs from UAB

Advisory Committee Chair

Elizabeth Sztul

Advisory Committee Members

James Collawn

Anne Theibert

Qin Wang

Bradley Yoder

Document Type

Dissertation

Date of Award

2012

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Vesicular transport is an essential cellular process that facilitates the movement of molecules within a cell. The importance of vesicular transport is highlighted by numerous human diseases that result from mutations affecting protein trafficking. The Brefeldin A-inhibited guanine nucleotide exchange factor 2 (BIG2) has emerged as a key regulator of protein trafficking within the late secretory pathway, between the trans-Golgi Network (TGN) and endosomal compartments and within the recycling endosomal pathway. Mutations in BIG2 lead to Autosomal Recessive Periventricular Heterotopia with Microcephly (ARPHM), a disorder characterized by severe brain malformations. BIG2 stimulates nucleotide exchange of GDP for GTP on ADP-ribosylation factors (ARFs). Once GTP-bound, ARFs become active and initiate a cascade of events that initiate vesicle formation and transport of proteins between compartments. BIG2 is a soluble cytoplasmic protein that must be recruited to specific sites at the membrane of the trans-Golgi network (TGN) to activate ARFs, but it is currently unknown how BIG2 is targetted to the TGN. A key goal of this study was to explore membrane association of BIG2. We found that membrane association of BIG2 was regulated through the enzymatic activity of another guanine nucleotide exchange factor called GBF1. GBF1 functions to regulate protein traffic within the early secretory pathway, at the ER-Golgi interface. The regulation of BIG2 recruitment by GBF1 may serve to coordinate early (ER-Golgi) and late (Golgi-TGN) traffic events to ensure efficient trafficking of secretory cargo through these compartments. The GDP/GTP exchange activity of BIG2 resides within a conserved Sec7 domain and is specific for ARF1 and ARF3. Crystallographic analyses and structure-function studies of the Sec7 domain have provided basic mechanistic insight into the exchange reaction, but the mechanisms responsible for substrate specificity of BIG2 remain unknown. We found a novel regulatory motif within the C-terminus of the Sec7 domain in BIG2 essential for ARF binding and nucleotide exchange. A loop after the J-helix (loop>J) is predicted to make contacts with ARFs and might participate in the recognition and binding of specific ARFs. These novel findings provide new insights into regulatory mechanisms that facilitate membrane association of BIG2 and control the selectivity of its enzymatic exchange activity.

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