טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentRawet-Slobodkin Moran
SubjectCharacterization of Determinants in ArfGAP1 Mediating its
Function in the COPI System of Vesicular Traffic
DepartmentDepartment of Biology
Supervisor Professor Emeritus Dan Cassel
Full Thesis textFull thesis text - English Version


Abstract

Membrane traffic mediated by COPI vesicles is regulated by the small G-protein Arf1. Membrane bound Arf1-GTP recruits the heptameric coat complex coatomer, promoting vesicle budding and cargo selection. Shedding of the coat which is a prerequisite for vesicle fusion depends on GTP hysrolysis on Arf1, catalyzed by the Golgi associated GTPase activating proteins (ArfGAPs).

In this work, we have investigated functional determinants in the non-catalytic part of ArfGAP1 that mediate its Golgi targeting and interaction with coatomer.

We identified a short stretch in which several hydrophobic residues contribute to Golgi localization of ArfGAP1. Mutations in this region strongly diminished Golgi localization, and also abrogated the in vitro activity of ArfGAP1 on Arf1. In addition, a major coatomer-binding determinant at the C-terminus of ArfGAP1 was identified. This determinant interacted with coatomer through the δ-subunit. Evidence for a role of the carboxy determinant in ArfGAP1-coatomer interaction in vivo was provided by a reporter fusion assay designed to identify functional determinants in cytosolic trafficking proteins. In this assay, the protein under study is fused to CD4, a type I protein that is normally localized to the plasma membrane. A diversion of CD4 localization to the ER is taken as indication for signals on the fused protein that recruit components of the COPI machinery which would mediate its Golgi to ER retrieval. The carboxy diaromatic motif that we identified in ArfGAP1 was required for ER diversion of the CD4 fusion.

Attempts to demonstrate that CD4 fusions cycle between the ER and the Golgi were unsuccessful. CD4 fusions were strongly localized to the ER in cells even under treatments known to trap cycling proteins in the ER-Golgi intermediate compartment. This led us to explore the issue of retrieval vs. retention in a broader manner, employing both CD4 and VSVG/ts045 as reporter proteins and extending the study to include carboxy dilysine signals which are thought to mediate protein retrieval to the ER through COPI vesicles. We found that VSVG/ts045 fused to coatomer interacting signals was ER localized, but could exit the ER. Moreover, by following the transport of VSVG fusions we found that different coatomer binding dilysine signals had different effect on the transport of VSVG which correlated with their ability to interact with coatomer. Our results indicate that localization/transport of membrane proteins in the early secretory pathway relies on multiple elements which determine the relative efficiency of ER exit and ER retrieval.