The synthesis and proteasomal degradation of a model substrate Ub5DHFR.

The importance of substrate polyubiquitination in protein degradation has been established for many years. However, the many intricacies of substrate recognition and ubiquitination by E3 enzymes have greatly limited access to degradable substrate in vitro. Thus, detailed analysis of protein degradation using purified 26S proteasomes has been difficult. The ability to synthesize polyubiquitin chains in a test tube has provided a method to make large quantities of a specific polyubiquitinated substrate, Ub(5)DHFR. This chapter focuses on the synthesis and degradation of this model substrate.