RECONSTITUTING UBIQUITINATION REACTIONS WITH AFFINITY-PURIFIED COMPONENTS AND P-32 UBIQUITIN

The discovery of protein ubiquitination in a broad range of organisms and experimental settings has raised the need for a straightforward wa y to characterize the mechanism of substrate targeting, using purified components. The mechanism of ubiquitin conjugation to proteins has be en extensively studied and is mediated by a family of evolutionarily c onserved proteins, We have used previously described expression system s to purify the relevant targeting components of the ubiquitin system. These methods yielded substantial amounts of highly purified and cata lytically active enzymes that permitted their use in reconstituting pr otein ubiquitination. We monitored ubiquitination reactions with P-32- ubiquitin rather than I-125-ubiquitin. This advance makes the procedur e accessible to a broader range of experimentalists, since it eliminat es the additional concerns involved in handling I-125-isotope. Further more, the strategies described here can be used to investigate the eff ects of specific mutations introduced into ubiquitin or the targeting components (El, Ubc/E2, and E3) of this pathway. (C) 1998 Academic Pre ss.