Ubiquiton – an inducible, linkage-specific polyubiquitylation tool

Here we introduce a set of engineered ubiquitin protein ligases and matching ubiquitin acceptor tags for the rapid, inducible linear (M1-), K48-, or K63-linked polyubiquitylation of proteins in yeast and mammalian cells. By applying the so-called ‘Ubiquiton’ (Ubo) system to proteasomal targeting and the endocytic pathway, we validate this tool for soluble cytoplasmic and nuclear as well as chromatin-associated and integral membrane proteins and demonstrate how it can be used to control the localization and stability of its targets. SILAC-based and label-free quantitative proteomics experiments were performed to verify the linkages of the polyubiquitin chains produced on a model substrate, GFP, in yeast and to identify potential off-target effects of a transgenic E3 system in human cells.