Synchronizationof Secretory ProteinTraffic in Mammalian Cells
The journey ofsecretory proteins, from their synthesis in the ER totheir arrival in their target compartment (e.g. the plasmamembrane, the extracellular space or the lysosomes)can take many paths. Understanding how a protein traffics through thesepathways is key to understanding, and potentially perturbing, its function. Inorder to dissect protein traffic in different systems, Franck Perez’slab, from the Institut Curie in Paris,has developed the retentionusing selecting hooks (RUSH) system.
How does it work? RUSHis an ingenious two-state assay based on the reversible interaction between ahook protein stably localized in the donor compartment (e.g. the ER) and areporter protein of interest (Figure A). Franck Perez’s lab has engineered hookproteins fused to a streptavidin core that are able to retain reporter proteinsfused to the streptavidin-binding peptide (SBP). The addition of biotindisrupts this interaction and thus triggers a synchronous release of thereporters which can then be tracked along the secretory pathway (Figure B). TheRUSH system has already been used to study transport characteristics of variousGolgi and plasma membrane reporters as well as secretory proteins or proteinstargeted to membrane sub-domains. It has also been used to observe intra- andpost-Golgi segregation of cargo during their transport. Finally, the system canbe adapted to cellular screening to identify molecules that can perturb proteintransport.
Franck Perez’s lab hasdeveloped a whole collection of hook and reporter proteins that can be used to test diversesecretory routes in various conditions. The plasmids and lentiviral vectorsencoding these proteins can also be used with your own reporters to deciphertheir trafficking routes. This collection is now available at Addgene, so onyour marks...get set...RUSH.