|Ph.D Student||Levi-Tal Sharon|
|Subject||Golgi-Associated ArfGAPs: Characterization of Targeting|
Determinants and Interaction Partners
|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 as well as intra-Golgi transport. COPI vesicle biogenesis is controlled by the cytosolic factor Arf1, a small GTPase of the Ras superfamily. After the formation of a COPI-coated vesicle GTP hydrolysis on Arf1, catalyzed by Arf1 GTPase-activating proteins (ArfGAPs), promotes coat release and allows vesicle fusion with the target membrane. Mammalian cells express three Golgi-localized ArfGAPs, ArfGAP1-3. These proteins consist of an N-terminal catalytic domain and a non-catalytic part which is characterized by high proportion of intrinsically unstructured sequences.
In the first part of this work we have characterized motifs in ArfGAP1 termed ALPS. Previous work has shown that an ALPS motif in ArfGAP1 folds into a helix upon binding to highly curved membranes and is required for its Golgi targeting. In work done in collaboration with Antonny’s group we identified of a second ALPS motif in ArfGAP1, termed ALPS2, and showed that this motif plays a role in Golgi targeting of the protein. We subsequently studied in detail the topology of the two ALPS motifs in ArfGAP1 and its brain isoform. These structure-function investigations revealed that the two ALPS motifs comprise together a region of approximately 100 amino acids, and are separated by a very short break.
In the second part of this study we identified novel interaction partners of Golgi ArfGAPs by using pulldown experiments and mass spectrometry analysis. These novel interactors included clathrin and its adaptors AP-1 and AP-2. Truncation and mutational analysis led to the identification of several di-aromatic adaptor-binding motifs within the non-catalytic part of ArfGAP1. Two such motifs interacted with AP-1 and AP-2, whereas a carboxy terminal motif was identified as a major coatomer-binding determinant and was found to interact with coatomer through the δ-subunit. We also looked for interactions of ArfGAP1-3 with components of the fusion machinery, the SNARE proteins. We found that the mammalian v-SNARE GS27 interacts specifically with the Golgi ArfGAPs, and that this interaction is mediated through the ArfGAP catalytic domains and the Habc helix of GS27.
The results of this work led to new insight on the mechanism mediating the Golgi targeting of ArfGAP1 and its interaction with coat and coat adaptors. Additionally, we have identified novel interaction of the three Golgi- associated ArfGAPs with the v- SNARE GS27.