|M.Sc Student||Volodin Alexandra|
|Subject||Studies of the Interaction between the Calcium-Binding|
Protein Secretagogin and Components of the COPI
|Department||Department of Biology||Supervisor||Professor Emeritus Dan Cassel|
|Full Thesis text|
The secretory pathway of mammalian cells mediates vesicle-mediated transport of proteins among different cellular compartments. COPI vesicles mediate retrograde transport from the Golgi to the ER as well as intra-Golgi transport. The generation of COPI carriers is regulated by small G proteins of the Arf family. Arfs are recruited to the Golgi upon GTP binding, whereupon they recruit coatomer to the membrane. Following vesicle budding the vesicle become uncoated in a process that depends on GTP hydrolysis on Arf, triggered by ArfGAP proteins. There are three Golgi-associated ArfGAPs (ArfGAP1-3), which all present sites for interaction with coatomer and have amphipathic stretches for Golgi localization. ArfGAP2/3 are closely related ArfGAPs, while ArfGAP1 belongs to a different ArfGAP class. It was recently reported that ArfGAP2/3 interact with the calcium-binding protein secretagogin (SCGN). SCGN is expressed in neuroendocrine cells and interacts with several trafficking proteins in vitro; however, the role of these interactions is currently unknown.
The aim of this study was to characterize mode of interaction of SCGN with Golgi ArfGAPs and find its possible role in the COPI system. Testing the specificity of the interaction of SCGN with Golgi ArfGAPs, we found efficient interaction with ArfGAP2/3 but no interaction with ArfGAP1. Truncation analysis revealed that there are two sites of SCGN interaction on the non-catalytic part of ArfGAP3. One site is located between aa 267-361 and the other site is a carboxy amphipathic motif, previously shown to be required for ArfGAP3 Golgi localization. The ArfGAP3-SCGN interaction was Ca-dependent (EC50 ̴ 100 µM). Somewhat unexpectedly, overexpression of SCGN had no significant effect on Golgi interaction of either ArfGAP3 or coatomer. We considered that these results might be due to the low levels of calcium prevailing in cytosol which prevent efficient SCGN-ArfGAP3 interaction. We tested this possibility by increasing the cytosolic Ca2 concentration using a Ca ionophore. Unexpectedly, we found that the Golgi localization of ArfGAP2/3 was disrupted under these conditions, thus preventing an assessment of SCGN effects at elevated cytosolic Ca2 concentrations. The mechanism of the Ca2 effect on ArfGAP2/3 Golgi localization will be the subject of future studies.