|Ph.D Student||Iliana Edith Szafer Glusman|
|Subject||Role of ArfGAP1 in the COPI Transport System|
|Department||Department of Biology||Supervisor||Professor Emeritus Cassel Dan|
The formation of COPI transport vesicles is initiated by the small GTPase Arf1 which cycles on and off the membrane coupled to cycles of GTP binding and hydrolysis. In its GTP bound form Arf1 acts to recruit the coat protein complex (coatomer) to the Golgi membrane. The binding of coatomer to the membrane causes the membrane to deform into a bud, to which cargo proteins are incorporated. Vesicle budding is followed by GTP hydrolysis-mediated uncoating, a pre-requisite for vesicle fusion. A family of GTPase activating proteins (GAPs) catalyzes GTP hydrolysis on Arf1. The activity of these proteins needs to be strictly regulated for adequate vesicle formation and consumption. Previous studies in well-defined systems indicated that the activity of Arf GAP1, a mammalian GAP that acts at the Golgi, is regulated by membrane lipids. More recent studies that used a soluble Arf1 mutant demonstrated that GTP hydrolysis may be strongly stimulated by coatomer. In this work we show that coatomer stimulation of GAP activity on the soluble mutant Arf1 does not occur in the presence of a different member of the ArfGAP family, or in a more physiological system that measures GTP hydrolysis on wild type Arf1 that is bound to liposomes. Using an assay that measures GAP activity at the Golgi membrane we revealed that ArfGAP1 activity is stimulated by coatomer by less than two fold, and the same is true for the overall activity of Arf GAPs that are present in brain cytosol. These findings suggest that in the biological membrane, proximity between Arf1 and its GAP generated by their mutual binding to membrane phospholipids may be sufficient for stimulation of Arf1 GTPase activity, and coatomer is not essential for GAP activity as previously suggested. However, coatomer may further modulate GAP activity in order to fine-tune the vesicle budding process.
GTP hydrolysis catalyzed by GAP was recently suggested to mediate sorting of cargo proteins into COPI vesicles. In addition, the cytoplasmic tail of p241, a protein that is present in both anterograde and retrograde ER to Golgi vesicles, was reported to inhibit GTP hydrolysis on a soluble Arf1 mutant. In this work we found that p241 directly inhibits Arf GAP1 activity measured on synthetic liposomes and on the Golgi membrane, suggesting a direct effect of p241 on GAP-mediated GTP hydrolysis. Experiments with synthetic peptides revealed that an inhibitory motif includes a tyrosine and a diphenylalanine within the cytosolic tail of the protein. We propose that proteins such as p241 act to coordinate cargo sorting with the formation of the COPI vesicle.