|M.Sc Student||Pevzner Irit|
|Subject||Characterization of the Interaction between the COPI|
Regulators ArfGAP2/3 and the Gamma Subunit of the
|Department||Department of Biology||Supervisor||Professor Emeritus Dan Cassel|
|Full Thesis text|
Transport of membranes and proteins in eukaryotic cells is mediated mainly by vesicular carriers. COPI coated vesicles mediate retrograde transport from the Golgi to the ER and intra-Golgi transport.
The cytosolic precursor of the COPI coat is a conserved heptameric complex called coatomer consisting of seven subunits: a-, b-, b ' -, g-, d-, e- and z-COP. In higher eukaryotes there are two isoforms of the coatomer g- and z-COP subunits. In mammalian cells the presence of three coatomer isoforms (g1z1, g2z1 and g1z2) with statistically significant segregation within the Golgi apparatus was observed, suggesting that different isoforms of coatomer might have differential preferences in their interactions with cargo and accessory proteins.
The formation of COPI vesicles is initiated by the small GTPase Arf1. In its GTP-bound form, Arf1 acts to recruit coatomer to the Golgi membrane. GTP hydrolysis on Arf1 promotes coat release and allows vesicle fusion with the target membrane. The exchange of GDP for GTP on Arf1 depends on a guanine exchange factor (GEF), whereas GTP hydrolysis depends on a GTPase activating protein (GAP). Work in our group focuses on the role of the ArfGAPs in the COPI system.
Mammalian cells express three Golgi-localized ArfGAPs, ArfGAP1, ArfGAP2 and ArfGAP3. ArfGAP2 and ArfGAP3 interact with coatomer g-COP subunit and their Golgi membrane association depends on this interaction. Recent work at our lab revealed an evolutionary conserved stretch in the middle of ArfGAP2/3 as a critical determinant mediating coatomer and g-COP appendage binding. It has previously been reported that the g-COP appendage domain possesses one binding site, comprising small hydrophobic pocket, for interaction with ArfGAP2/3. However these findings are incompatible with the identification in our lab of a 40-50 residue-long ArfGAP2/3 stretch that is responsible for the interaction with the g-COP appendage domain. Thus additional binding site(s) for ArfGAP2/3 on the g-COP appendage domain must exist.
This work reveals that the g-COP appendage domain can interact with ArfGAP2/3 at a newly identified binding site, which consists of a relatively large hydrophobic pocket. Mutations at this pocket abrogated the binding of ArfGAP2/3 in vitro and in vivo. We further provided evidence that both g-COP isoforms participate in similar cellular pathways and both the g1 and g2 appendage domains were capable of interacting in vitro with ArfGAP2 and ArfGAP3, although with differential efficiently, and both could with ArfGAP2 in transfected cell assays.