טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentDegtyar Elena
SubjectFunctional Interaction between Coat Proteins and the Arf1
Regulator ArfGAP1
DepartmentDepartment of Biology
Supervisor Professor Emeritus Dan Cassel


Abstract

The G protein Arf1 regulates the formation of coated vesicles through its GTPase cycle. The removal of the coat depends on the hydrolysis of GTP, which requires a GTPase-activating protein (GAP). Our lab is studying a Golgi-localized GAP termed ArfGAP1. Previous studies showed that the coat of the Golgi-derived vesicles (coatomer) stimulates ArfGAP1 activity under certain conditions. The goal of this work was to investigate AP-1, a component of TGN-derived clathrin coated vesicles, participates in ArfGAP1 regulation. We developed an in vitro system that allowed testing ArfGAP1 regulation in the presence of liposomes. Under these conditions AP-1 was able to stimulate the activity of the catalytic fragment of ArfGAP1 (GAP136). This stimulation depended on the presence of peptides representing a tyrosine sorting signal. Subsequent experiment revealed that unlike coatomer, AP-1 stimulated not only GAP136 activity, but also that of the full length ArfGAP1. In addition, clathrin was found to inhibit AP-1 - dependent stimulation of GAP activity. Immunofluorescence labeling revealed that AP-1 did not co-localize with either ArfGAP1 or Arf1. Our findings suggest that AP-1 and clathrin play a role in the regulation of GTP hydrolysis on Arf1 during the cycle of vesicle formation and uncoating. However, based on the immunofluorescence experiments, the ArfGAP responsible for GTP hydrolysis might not be ArfGAP1. Taken together with previous studies, the regulation of GTP hydrolysis by coat proteins now appears as a common denominator in all small G protein-dependent coated vesicle systems.