|Ph.D Student||Lifshitz Lena|
|Subject||Dissecting the Function of the ArfGAP2/3 Proteins in the|
COPI Trafficking Machinery
|Department||Department of Biology||Supervisor||Professor Emeritus Dan Cassel|
|Full Thesis text|
The COPI system is among the major routes of vesicular trafficking between membrane-bound compartments of eukaryotic cells. The cytosolic precursor of the COPI coat is coatomer, a conserved protein complex consisting of seven subunits. The formation of COPI vesicles is initiated by the small GTPase Arf1, which membrane association/dissociation cycle is coupled to GTP binding and hydrolysis. The COPI system is regulated by two types of ArfGAPs, ArfGAP1 and the closely-related ArfGAP2 and ArfGAP3. Besides the amino-terminal catalytic domains these two types of ArfGAPs do not share any significant sequence similarity, although in both the non-catalytic parts are largely unstructured intrinsically. Whereas ArfGAP activity and localization are regulated through its membrane interacting ALPS (amphipathic lipid packing sensor) motifs, less is known about mechanisms regulating ArfGAP2/3.
In the first part of this work we employed a modification of the reporter fusion approach to identify several functional regions in ArfGAP2/3. Two of these determinants -central basic stretch and a carboxy-amphipathic motif- were found to be required for Golgi targeting of the proteins. Pull-down assays showed that the basic stretch interacts directly with coatomer. Moreover, coatomer was found essential for the catalytic activity of ArfGAP2/3, and the effect of coatomer depended on its interaction with the basic stretch. Like the ALPS motifs, the carboxy motif of ArfGAP2/3 could fold into a helix in the presence of small liposomes; however, this motif had only a minor role in the regulation of ArfGAP2/3 activity.
In the second part of this study we characterized the interaction of ArfGAP2 and ArfGAP3 with particular coatomer subunits. We confirmed a previous report that ArfGAP2 but not ArfGAP3 interacts strongly with an appendage domain of g-COP subunit, We showed that the basic stretch identified in the first part of this work mediates ArfGAP2 interaction with an appendage domain of g-COP subunit. Using yeast two-hybrid method we identified novel interaction of ArfGAP3 with the b subunit of coatomer. We found that a region encompassing residues 300-360 in ArfGAP3 (“the B region”), initially identified by the reporter fusion assay, plays a role in this interaction.
The results of this work led to insights on previously unknown mechanism mediating the Golgi targeting and the regulation of catalytic activity of ArfGAP2/3. Further biochemical analysis together with advanced structural studies will be required to further understand the role of distinct elements on ArfGAP2 and ArfGAP3 in the cellular functioning of the proteins.