Nature Methods

A new system for the in vivo tracking of protein ubiquitination states offers considerably more detailed kinetic information than earlier techniques, providing researchers with a real-time chronicle of protein modification rather than a mere snapshot.

Ubiquitination, the enzymatic tagging of a protein with one or more copies of the 76-aa protein ubiquitin, has long been perceived as the biochemical equivalent of posting a "Condemned" sign, dooming the recipient to rapid degradation. However, recent evidence shows that this modification need not be a death sentence, and can play an important part in a variety of protein functions and signaling pathways. As scientists come to grips with a broadening range of ubiquitin-modulated activities, interest has grown in developing accurate and non-disruptive methods for tracking dynamic changes in ubiquitination levels.

In the December issue of Nature Methods, a team of Canadian and French researchers presents a novel strategy for the real-time detection of protein ubiquitination in living cells. Their system relies on a technique called BRET (bioluminescence resonance energy transfer), wherein a luminescent energy _donor_ molecule transfers energy to a proximal _acceptor_ molecule, which responds by fluorescing at a distinctive wavelength. In this particular approach, a protein of interest fused to luciferase is co-expressed with a GFP-ubiquitin fusion—if ubiquitination occurs, a readily detectable BRET signal is produced. This system proved capable of revealing the distinctive kinetics of ubiquitination for different proteins, and even detecting protein-protein interactions concomitant with an ubiquitination event. Importantly, this method also offers the possibility of monitoring highly transient ubiquitination changes, modifications that are considerably more difficult to observe by conventional immunochemical approaches. In an accompanying News and Views piece, noted signal transduction researcher Robert Lefkowitz concludes, "It seems likely that BRET assays such as those described by Perroy et al. will greatly expedite the unraveling of the biological roles of... post-translational modifications related to ubiquitination."