טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentLior Regev
SubjectArfGAP1 - Interaction with the Golgi Apparatus and
Characterization of New Splice Variants
DepartmentDepartment of Biology
Supervisor Professor Emeritus Cassel Dan


Abstract

The COPI system mediates vesicular transport in the ER-Golgi shuttle. The formation of COPI carriers is triggered by the conversion of the small GTPase Arf1 to its GTP-bound form. The hydrolysis of GTP, which constitutes a prerequisite for coat dissociation and vesicle fusion, depends on the action of the Arf-directed GTPase-activating protein ArfGAP1. We have used two COPI-affecting drugs, Brefeldin A (BFA) and fluoride ions, for addressing the question of whether ArfGAP1-Golgi interaction is mediated through an Arf1-dependent or independent mechanism. We show that in BFA treated cells, endogenous ArfGAP1 presents similar kinetics to that of coatomer, whereas a GFP fusion of the non-catalytic domain of ArfGAP1 presents a much slower kinetics. Fluoride ions are known to cause a stable binding of coatomer to the Golgi. Our findings show that fluoride ions cause the disruption of the Golgi localization of ArfGAP1. Based on these findings, we suggest that ArfGAP1-Golgi interaction is mediated through its non-catalytic domain in an Arf1-independent way, as well as through Arf1-dependent interaction through the catalytic domain.

In a second project we report the identification of two novel tissue-specific ArfGAP1 splice variants, containing a small in-frame deletion and/or insertion within the non-catalytic domain. One of the new variants constitutes the major ArfGAP1 form in brain tissue, whereas the other is a minor form in heart. Expression of GFP fusion of the variant ArfGAP1 non-catalytic domain revealed Golgi localization, similar to the originally-identified form. Studies are in progress in attempts to understand the role of the variant form in brain.