Reconstituting ubiquitination reactions with affinity-purified components and ³²P-ubiquitin

The discovery of protein ubiquitination in a broad range of organisms and experimental settings has raised the need for a straightforward way to characterize the mechanism of substrate targeting, using purified components. The mechanism of ubiquitin conjugation to proteins has been extensively studied and is mediated by a family of evolutionarily conserved proteins. We have used previously described expression systems to purify the relevant targeting components of the ubiquitin system. These methods yielded substantial amounts of highly purified and catalytically active enzymes that permitted their use in reconstituting protein ubiquitination. We monitored ubiquitination reactions with ³²P-ubiquitin rather than ¹²⁵I-ubiquitin. This advance makes the procedure accessible to a broader range of experimentalists, since it eliminates the additional concerns involved in handling ¹²⁵I-isotope. Furthermore, the strategies described here can be used to investigate the effects of specific mutations introduced into ubiquitin or the targeting components (El, Ubc/E2, and E3) of this pathway.