Monitoring of ubiquitin-dependent proteolysis with green fluorescent protein substrates.

A reliable and robust means of evaluating the functional status of ubiquitin-dependent proteolysis in living cells is to follow the turnover of readily detectable reporter substrates. During the past few years, several reporter substrates have been generated by use of the green fluorescent protein (GFP), which is converted for this purpose from a normally very stable protein into a short-lived substrate of the ubiquitin/proteasome system. These short-lived substrates are valuable tools providing researchers with unique information about the absence or presence of blockades in this system in living cells. We have recently generated the first transgenic mouse model for monitoring the ubiquitin/proteasome system based on the ubiquitous expression of a GFP-based proteasome substrate. Together these models can be used to study ubiquitin-dependent degradation in health and disease and for the identification of small synthetic compounds or proteins capable of modifying the activity of the system. In this chapter, we describe the basic principles of GFP-based reporter substrates, their strengths and weaknesses, and a number of protocols that can be used to study the ubiquitin/proteasome system in yeast, cell lines, and transgenic mice.