|M.Sc Student||Abraham Omer|
|Subject||Regulation of intracellular trafficking by|
masking/unmasking of targeting signals
|Department||Department of Biology||Supervisor||Professor Emeritus Dan Cassel|
|Full Thesis text|
The Nobel laureate Günter Blobel conceived the idea that proteins incorporate intrinsic address labels that govern their transport and guide them to their destinations in the cell. Systems that allow the control of transport though synchronization of cargo flow have been valuable in elucidating the underlying trafficking mechanisms, a task which can be met with only limited success when the cell is at steady state. Classical methods such as temperature blocks or the thermosensitive protein ts045-VSVG which are employed to investigate the secretory system impose non physiological conditions on the cell. Most current approaches rely on ligand or light-controlled dimerization that either elicit or constrain the transport of a reporter. Recently, Boncompain et al. have presented a new synchronization approach which relies on selective retention and regulated release of cargo molecules from various donor compartments, while employing the interaction between streptavidin (SA) and streptavidin-binding peptide (SBP). However, this approach requires to design a tailored hook for each donor organelle and it cannot be applied to retrograde transport in the secretory system. I developed an alternative approach for transport regulation which is based on reversible masking of targeting signals, also relying on the SA-SBP interaction. The signal is generated within or immediately following the SBP sequence that is appended to the reporter. The binding of co-expressed SA to SBP causes signal masking, whereas addition of biotin, the natural ligand of SA, causes complex dissociation and triggers protein transport to the target organelle. By employing the SV-40 large T antigen nuclear localization signal (NLS) or a type 1 peroxisomal targeting signal (PTS1) along with a soluble reporter, I applied the masking/unmasking approach to the control of nuclear and peroxisomal protein import. To demonstrate biotin-dependent trafficking through the secretory pathway I combined a transmembrane reporter with an artificial endocytic acidic dileucine signal which was generated within the SBP sequence or a dilysine COPI retrograde signal. Additionally, by simultaneous masking of the COPI and endocytic signals, I was able to generate a synthetic pathway for efficient transport of a reporter from the plasma membrane to all the way to the ER. Introduction of a linker which separated the SBP from the NLS abrogated masking, thus supporting the idea that NLS masking results from steric hindrance between the SBP-bound SA and the NLS cognate receptor. Surprisingly, linker insertion did not affect masking in the case of the PTS1 and COPI dilysine signals, indicating that in these two cases masking is due to mechanisms other than steric interference with receptor binding. Be the mechanism as it may, masking of both PTS1 and COPI dilysine signals was efficient and reversible, making this approach useful for transport synchronization. This system operates under physiological conditions, displays a short response time and notably, overcomes the hurdle of monitoring retrograde transport mediated by COPI dilysine signals. After its successful implementation in multiple intracellular transport processes, the masking/unmasking approach is ready to be adopted to other systems as well and could answer the needs of additional researchers in the future.