Short-lived green fluorescent proteins for quantifying ubiquitin/proteasome-dependent proteolysis in living cells

The ubiquitin/proteasome-dependent proteolytic pathway is an attractive tar get for therapeutics because of its critical involvement in cell cycle prog ression and antigen presentation. However, dissection of the pathway and de velopment of modulators are hampered by the complexity of the system and th e lack of easily detectable authentic substrates. We have developed a conve nient reporter system by producing N-end rule and ubiquitin fusion degradat ion (UFD)-targeted green fluorescent proteins that allow quantification of ubiquitin/proteasome-dependent proteolysis in living cells. Accumulation of these reporters serves as an early predictor of G2/M arrest and apoptosis in cells treated with proteasome inhibitors. Comparison of reporter accumul ation and cleavage of fluorogenic substrates demonstrates that the rate-lim iting chymotrypsin-like activity of the proteasome can be substantially cur tailed without significant effect on ubiquitin-dependent proteolysis. These reporters provide a new powerful tool for elucidation of the ubiquitin/pro teasome pathway and for high throughput screening of compounds that selecti vely modify proteolysis in vivo.