טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentBen-Asher Noa
SubjectPost Translational Modifications in Arf-GAPs
DepartmentDepartment of Biology
Supervisor Professor Emeritus Dan Cassel
Full Thesis textFull thesis text - English Version


Abstract

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. A guanine nucleotide exchange factor (GEF) induces the exchange of bound GDP to GTP in Arf1 leading to its anchorage at the Golgi membrane, where it recruits the heptameric COPI coat complex (coatomer) to the membrane.  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, ArfGAP2 and ArfGAP3. These proteins consist of an N-terminal catalytic domain and a non-catalytic part that is characterized by high proportion of intrinsically unstructured sequences. Whether the activity of these ArfGAPs is subjected to regulation is unknown.

The COPI system has an important role in retrieving ER resident proteins, such as chaperons, that escape the ER unintentionally. Chaperons in the ER help newly synthesized proteins to reach their correct folded state, and prevent accumulation of unfolded or misfolded proteins in the lumen of the ER. The condition in which the accumulation of unfolded or misfolded proteins reaches a degree that exceeds the folding capability of ER chaperones, is termed ER stress. The cellular response to this stress is termed unfolded protein response (UPR). The connection between the COPI system and ER stress is not studied in the literature, and we sought to find such connection.

In the first part of this study, I investigated whether Golgi ArfGAPs are regulated by phosphorylation. I searched for phosphorylation sites in ArfGAP1,2,3 using mass spectrometry analysis, and compared them to an online database (phosphosite). I identified multiple, previously unknown phosphorylation sites in all three proteins. Several of these sites had phenotypic effect as revealed by phospho- mimetic mutations to aspartic acid: ArfGAP1 Y208D and S246D, as well as ArfGAP2 S494D and T508D had a negative effect on Golgi localization, while ArfGAP3 S428D appeared to abrogate coatomer interaction.

In the second part, I investigated the effect of a potential disruption of the proper cycling of the COPI system due to overexpression or silencing of ArfGAPs on ER stress. As an indicator to the levels of ER stress I measured the induction of UPR related genes by real time PCR. I found that overexpression of ArfGAPs, both wild type and catalytically inactive, did not affect UPR level. However, silencing of ArfGAP2 with siRNA induces UPR, in accordance with our hypothesis that disrupting activity of the COPI system may lead to ER stress due to a abrogation of chaperone retrieval to the ER. On the other hand, we found that silencing ArfGAP3 decreases UPR, in contrary to our hypothesis. Further study is needed to clarify the source of the different effects these two closely-related proteins have on ER stress.