All ETDs from UAB

Advisory Committee Chair

Elizabeth Sztul

Advisory Committee Members

James Collawn

Guillermo Marques

Anne Theibert

Anne Woods

Document Type

Dissertation

Date of Award

2009

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

The secretory pathway within a cell consists of series of membrane compartments connected by shuttling secretory vesicles containing cargo that travel from endoplasmic reticulum (ER) though ER-to-Golgi intermediate compartment (ERGIC), the Golgi apparatus, and endosomal compartment to the plasma membrane (PM). The Golgi specific brefeldin A (BFA)-resistant factor 1 GBF1 has been identified as an important factor involved in traffic between ER and Golgi and within the Golgi apparatus. GBF1 belongs to a family of guanine nucleotide exchange factors (GEFs) that stimulate the exchange of GDP for GTP on small GTPases, ADP-ribosylation factors (ARFs). The exchange of the nucleotide activates ARFs and activated ARFs interact with variety of effectors including coat proteins and lipid modifying enzymes. In order to initiate the coat protein complex I (COPI) recruitment cascade, GBF1 must associate with membranes, but the membrane dynamic of GBF1 and its role in Golgi biogenesis were unknown. A central focus of this study was to explore membrane association of GBF1, the molecular events facilitated by GBF1, and to determine how interaction with lipid flippases integrates GBF1 activity with other traffic events. We found that GBF1 cycles rapidly between the cytosol and membranes and is stabilized on membranes when in a complex with ARF-GDP. Using siRNA mediated depletion we showed that GBF1 is necessary for membrane association of COPI. Although a secretory pathway capable of trafficking of soluble proteins in GBF1-depleted cells is maintained, GBF1-mediated ARF activation and COPI recruitment is required for trafficking of transmembrane proteins. The GBF1 catalytic domain (Sec7) is sufficient to catalyze nucleotide exchange by itself; therefore it was highly probable that the remaining part of GBF1 plays a regulatory role. We generated GBF1 mutants impaired in binding to putative lipid flippase and explored its cellular behavior and influence on Golgi morphology. Our results suggest that these interactions may be important for the modulation of GBF1 activity. The findings presented here further describe GBF1as a key molecule required for Golgi biogenesis and cargo trafficking, and will serve as a paradigm for other GEFs.

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