Intracelluar localization of ubiquitin-proteasome dependent proteolysis – UPS-Imaging

A large and still rapidly expanding body of reports highlights the critical role of the Ubiquitin-Proteasome System (UPS) in cellular homeostasis. This system is a major contributor for intracellular proteolysis, and is now recognized as both a central cellular machinery required for appropriate regulation of most biological pathways, and a major potential target for therapeutic intervention. The classical view of UPS functioning is that its substrates are first marked by poly-ubiquitylation, a post-translational modification that targets them for degradation by the proteasome. However, it is now clear that this view is an oversimplification: first, ubiquitylation is a highly dynamic, reversible modification that can serve many other functions than targeting protein for proteasomal degradation; second, not all proteasome substrates require prior ubiquitylation to be recognized and degraded by the proteasome; third, the term 'proteasome' itself is in fact a generic term that defines a family of related complexes, the specific functions of which are not well understood. Among the many questions raised by the complexity of this system, an important issue, still largely open, is where in the cell are localized both the UPS components and the main events that lead to degradation of a given substrate. The goal of our project is to get a deeper insight into this issue, by investigating, using in vivo imaging and interaction techniques (coupled, when necessary, to biochemical approaches), the intracellular localization of the different proteasome complexes (i.e. the 20S proteasome bound to its different regulators) and of the ubiquitylated forms of two regulators of cell proliferation, the proteins p53 and Cyclin A (CycA). This proposal exploits the complementary experiences of two groups from Montpellier (Olivier Coux (OC), CRBM-CNRS, and Jean-Marie Blanchard (JMB), IGMM-CNRS) in UPS analysis and in vivo interaction studies, respectively. In addition, the state-of-the-art 'Montpellier RIO Cell Imaging' facility, located in the CRBM, will provide an exceptional technical environment for the realization of our objectives. As recent results from our laboratories demonstrate the power and the fertility of such approaches, we are convinced that this project will lead to exciting new insights into the regulation and the mechanisms of action of the UPS.