|M.Sc Student||Karnit Gotliv|
|Subject||A Masking/Unmasking Approach for the Synchronization of|
COPI-Mediated Retrograde Golgi to ER Traffic
|Department||Department of Biology||Supervisor||Professor Emeritus Cassel Dan|
|Full Thesis text|
Endoplasmic reticulum (ER) resident proteins that have accidentally escaped from this organelle are retrieved from Golgi/ERGIC compartments by COPI system. However, retrograde COPI transport is hard to follow due to continuous cycling and the predominant ER localization of those residents. Synchronization of transport is instrumental in studying transport routes, allowing a kinetic follow-up by live cell imaging, differentiation between parallel transport routes etc. Recently, a new system for synchronization was developed, based on streptavidin (SA) and streptavidin-binding protein (SBP) interaction. This approach relies on a reversible association between a "hook" protein, fused to SA, and the protein of interest bearing SBP tag. Upon biotin addition, the protein of interest dissociates from the "hook" and travels to its target compartment synchronously. However, this system isn't applicable for studying retrograde trafficking. The aim of my study was to design a modification of the SA-SBP system to allow synchronized COPI-mediated transport of K(X)KXX signal tagged proteins. This approach is based on reversible signal masking, results in ER exit followed by unmasking using biotin, which binds SA and causes SBP dissociation. The signal is created immediately following SBP at the C-terminus of a reporter protein; we assumed that SA binding to SBP would mask the dilysine signal due to steric interference of the large COPI-coat binding. To follow retrograde transport from post-ER compartments, we used as a reporter the temperature-sensitive VSVG mutant that is ER-accumulated at 40°C, and is exported out of the ER as a synchronous wave at 32°C. The VSVG construct can then be accumulated in early secretory compartments, and subsequent unmasking with biotin should initiate retrograde transport. We demonstrate this approach to generate a regulated Golgi to ER transport using accumulation of the construct at the Golgi following over-night incubation at 40°C. Golgi accumulation was achieved using two different approaches. One approach was based on temperature blocking at 20°C, which inhibits vesicle budding from the trans-Golgi network, and the other made use of the photo-convertible dendra2 protein to selectively switch the fluorescence frequency of proteins that have reached the Golgi. By either approach, biotin addition led to the disappearance of the VSVG construct from the Golgi and its appearance at the ER, while in the absence of biotin the construct reached the plasma-membrane. With the ability to demonstrate retrograde transport, the masking-unmasking approach serves as a novel technique for demonstrating Golgi to ER transport and will be presented in this work.