| dc.description.abstract | COPI plays important roles in the cellular trafficking system by retrieving protein cargoes at the Golgi and endosomes. The COPI budding and fusion cycle starts when a small GTPase, Arf1, is recruited to the membrane. ArfGAP proteins regulate the COPI coat, but the molecular details of COPI recognition by ArfGAP proteins are still unclear. Here, by utilizing various biochemical and biophysical approaches, we were able to identify Arf1 and the yeast ArfGAP, Glo3, as direct binding partners of β’-COP, and reveal Glo3 associates with COPI through its interaction with β’-COP. Through ITC (Isothermal Titration Calorimetry) experiments and a structure-directed mutagenesis approach, we quantified and investigated the molecular basis of the interaction between β’-COP and the Glo3 BoCCS (Binding of Coatomer, Cargo, SNAREs) region. When tested in vivo, the loss of β’-COP/Glo3 interaction caused aberrant Golgi morphology and Ste2 mis-sorting to the vacuole, suggesting the β’-COP/Glo3 interaction is important for cargo recycling via the TGN and the endosomes. In addition, a new X-ray crystal structure of the Glo3 GAP domain was determined at 2.1 Å. The structure reveals a Cys4-family zinc finger motif with an invariant residue (R59) positioned to act as an “arginine finger” during catalysis. Comparisons among eukaryotic GAP domains show a key difference between ArfGAP1 and ArfGAP2/3 family members in the final helix located within the domain. Conservation at both the sequence and structural levels suggest the Glo3 GAP domain interacts with yeast Arf1 switch I and II regions to promote catalysis. Together, the data and structural model provide evidence for the Glo3 niche model within the assembled COPI coat and suggest its important roles in regulating the COPI though the molecular platform of β’-COP. | |
