Protein ubiquitylation is a modification to cellular protein factors that modulates their localisation, interactions or stability. It impacts on virtually every biological pathway, and its deregulation causes various diseases. Ubiquitin signalling is predominantly mediated by polymeric ubiquitin chains of distinct linkages. Yet, the relevance of these linkages is poorly understood, largely due to a complete lack of methods for direct manipulation of this feature. We have now designed an inducible system for the substrate- and linkage-selective polyubiquitylation of any target protein in vitro or in vivo. This tool, which we have named the “Ubiquiton” system, is based on a set of recombinant, tailor-made, linkage-selective ubiquitin ligases in combination with appropriate tags appended to the desired substrate proteins. We have optimized the enzymes and the targeting to their substrates in vitro and have designed expression constructs for budding yeast and human cells. In several instances, activity in cells has observed, while in other cases, further optimization is needed. Once this is complete, we will make the system available to the scientific community as an innovative research tool for investigating ubiquitin chain linkage in cells, the preparation of polyubiquitylated proteins for biochemical analysis in vitro, the identification of linkage-specific interaction partners and the control of protein fate in living cells. The Ubiquiton system will thus help researchers tackle fundamental questions about the functions of polyubiquitylation in health and disease.
