Single-molecule methods for measuring ubiquitination and protein stability

The ubiquitin-proteasome system (UPS) contributes to changes in cell state and homeostatic maintenance in humans by modulating the stability of about a third of human proteins. For example, cell-cycle regulation requires a central ubiquitin ligase, the anaphase-promoting complex/cyclosome (APC/C), which starts a ubiquitination cascade leading to the degradation of multiple targets. This targeted degradation is mediated by the 26S proteasome, a 2.5-MDa protein complex, which recognizes and degrades ubiquitinated proteins at rates partially controlled by the variations in ubiquitin chain topology. Substrate selectivity of ubiquitin ligases such as the APC/C and of the 26S proteasome from pools of near-identical targets reflects highly regulated kinetic mechanisms. Single-molecule techniques are powerful tools that allow distinction between differential substrate affinities and identification of reaction intermediates in complex mixtures. Here we describe fluorescence-based single-molecule imaging of in vitro ubiquitination reactions catalyzed by the APC/C and ubiquitin-dependent degradation reactions catalyzed by the 26S proteasome.