Advisory Committee Chair
Advisory Committee Members
Date of Award
Degree Name by School
Doctor of Philosophy (PhD) Heersink School of Medicine
All eukaryotic cells contain a secretory pathway composed of membrane-bound compartments connected by vesicles that transport cargo from the endoplasmic reticulum (ER) through the Golgi apparatus to various destination within and outside the cell. The Golgi Brefeldin A-resistant Factor 1 (GBF1) is required for protein traffic between ER and Golgi and within the Golgi. GBF1 belongs to a family of Guanine nucleotide Exchange Factors (GEFs) that stimulate the nucleotide exchange of GDP for GTP on small GTPases called ADP-ribosylation factors (ARFs). Once GTP-bound, ARFs become active and initiate a cascade of events that lead to vesicle formation. Thus, GBF1 is the upstream regulator that defines the site and timing of ARF activation and thereby controls vesicle formation and protein traffic. Yet, despite its critical role in cellular life, we remain ignorant how GBF1 itself is regulated in cells. Thus, we studied structure-function relationships within the non-catalytic domains of GBF1 to understand how they may regulate GBF1 activity. GBF1 is a large modular protein that exists as a homo-dimer, and we focused on the N-terminal Dimerization and Cyclophilin-Binding (DCB) domain and the C-terminal Homology Downstream of Sec7 (HDS3) domain. We engineered mutations in the DCB domain to disrupt GBF1 oligomerization and then used such mutant to assess the role of oligomerization in GBF1 function. My results show that oligomerization does not regulate GBF1 localization or catalytic activity, but impacts GBF1 stability. Thus, oligomerization regulates cellular function of GBF1 by regulating its degradation levels and thereby its cellular levels. We also introduced mutations within the most conserved regions of the HDS3 domain or removed the HDS3 and downstream region to study the role of HDS3 in GBF1 function. My results show that HDS3 mutants do not target to the Golgi and are functionally defective. Furthermore, HDS3 mutants exhibit altered folding of the N-terminus of GBF1, suggesting a model whereby the C-terminal HDS3 domain regulates the conformation of the N-terminal regions of GBF1 to modulate membrane association of GBF1. My findings provide novel insight into the role of the non-catalytic DCB and HDS3 domains in regulating protein levels and membrane association of GBF1.
Bhatt, Jay Manoj, "Structure-Function Relationships In The Sec7 Guanine Nucleotide Exchange Factor Gbf1" (2016). All ETDs from UAB. 1164.