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Abstract

Many fundamental discoveries in ubiquitin-proteasome research have relied on reconstitution of activities from purified or recombinantly expressed components. These include landmark discoveries of E1-E2-E3 mechanisms, in which ubiquitin (UB) is initially activated and then covalently shuttled between enzyme active sites and ultimately ligated to substrate or substrate-linked UBs during polyubiquitination. However, recent studies have unearthed enormous variations on the E1-E2-E3 theme; for example, one E3 may employ two distinct E2s, or two different E3s may act in a single assembly or in series, to prime substrates directly with UB and subsequently decorate them with myriad types of polyubiquitin chains. To dissect this complexity, it can be helpful to monitor specific UB transfer reactions in isolation, rather than the end-point products formed upon mixing all enzymes in a cascade. Pulse-chase assays enable observation of a single reaction step and also allow one to differentially label UBs carried by different enzymes within the same tube. In such assays, the "pulse" reaction generates a thioester-linked enzyme similar to UB intermediate, while the "chase" monitors UB transfer to downstream components over time. Here, we describe pulse-chase assays for detecting fluorescent-UB in E2 similar to UB intermediates. These assays enable direct assessment of particular ligation reactions, alone and in combination, to explore roles of multiple enzymatic cascades in the same tube. We anticipate this technique can be adapted to many different E2s, as well as thioester-forming E3s, to dissect ubiquitination by many distinct enzyme cascades.