Assay for proteasome-dependent protein degradation and ubiquitinated proteins

The ubiquitin-26S proteasome system (UPS) plays a crucial role in selective removal of short-lived target proteins, archiving fine-tuning of post-translation levels of the target proteins. Recently a number of ubiquitin ligases (E3) have been reported as essential regulators of various plant developmental cues and stress responses. To clarify the detailed biochemical and physiological function of the E3 proteins, identification of their target proteins is of great importance. A transient expression system with tobacco leaves is a powerful method to evaluate E3 function and target degradation via UPS. Here simple methods to assay proteasome-dependent protein degradation combined with a tobacco transient expression system and detection of accumulation of ubiquitinated proteins are presented.