טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentPevzner Masha
SubjectStudies of ArfGAP1 Function in Cellular Trafficking
Pathways
DepartmentDepartment of Biology
Supervisor Professor Emeritus Dan Cassel
Full Thesis textFull thesis text - English Version


Abstract

In eukaryotic cells, COPI coated vesicles mediate retrograde transport from the Golgi to the ER as well as intra-Golgi transport. COPI vesicle biogenesis is controlled by 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).  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.

In the first part of this study we searched for novel interactions between aromatic motifs on ArfGAP1 and trafficking proteins. Two tissue specific splice variants of ArfGAP1 from rat heart and brain were identified in our lab. The brain isoform contains an in-frame 10 residues insertion and a 22 residues deletion; within the insertion in this variant, an FW sequence was found critical for Golgi localization. We searched in databases for proteins interacting with Phe-Trp-comprising sequences and identified EPS15, a known regulator of transport in secretory pathways. Pull-down assay showed that the ArfGAP1 brain variant peptide does not interact with the EH3 domain of EPS15, but the ubiquitous ArfGAP1 peptide (that contains an FW sequence as well) does bind the EH3 domain. The residues Phe-Trp (aa 328-329) as well as a Phe (aa 332) were important for the interaction. Significantly, the same residues were found to form a motif for coatomer interaction.

In the second part, we investigated whether different ArfGAPs are recruited to the Golgi during synchronized cargo arrival. A previous study has reported that anterograde cargo transport causes recruitment of ArfGAP1 to the Golgi. The cargo employed was a temperature-sensitive VSVG mutant that is unfolded in 400C and kept at the ER. A shift to 320C allows folding of the VSVG and its transport through the Golgi to the plasma membrane (PM). We investigated whether in response to a wave of VSVG transport ArfGAP3 is recruited as well; however, no such recruitment was observed. While we could reproduce the experiment demonstrating ArfGAP1 recruitment, we found that this recruitment was due to the temperature shift rather than to VSVG transport, thus bringing the previously reported findings into question.