Background

Ubiquitination is a three-step process involving the sequential action of E1, E2, and E3 enzymes. In the first step, catalyzed by an E1 ubiquitin-activating enzyme, ubiquitin-adenylate is formed followed by ubiquitin transfer to a conserved cysteine on the enzyme. The second step involves a trans-thioesterification, in which the ubiquitin is transferred to a cysteine on the E2. In the third step, the E2, known as a ubiquitin-conjugating enzyme, cooperates with an E3 ubiquitin ligase to transfer ubiquitin to the target substrate, typically through the formation of an isopeptide bond to a lysine side chain (Zheng and Shabek, 2017). In many cases, target substrates can become poly-ubiquitinated through the attachment of further ubiquitin molecules. This attachment can take place on any of ubiquitin’s seven lysine side chains or on its amino terminal methionine, resulting in many permutations of ubiquitin conjugation (Zheng and Shabek, 2017). Specific ubiquitin linkages are associated with different cellular outcomes, although the understanding of this is incomplete (Komander and Rape, 2012). In an important “off-pathway” reaction, some E2s are also able to catalyze the formation of free di-ubiquitin, alone or with the assistance of a ubiquitin E2 variant (Wu et al., 2003; Wang et al., 2016; Burge et al., 2020).

The parasite Leishmania mexicana assumes different morphologies depending on its host: in the sand fly, it takes the motile promastigote form, whereas in a mammalian host, it takes the immotile amastigote form. Key to mammalian infection is the ability to undergo promastigote to amastigote differentiation. In L. mexicana, the E2 UBC2 and the ubiquitin E2 variant UEV1, which form a heterodimeric complex, are required for this process (Burge et al., 2020). Prior studies of the Saccharomyces cerevisiae UBC2 and UEV1 homologs UBC13 and MMS2, respectively, demonstrated that they could form K63-linked ubiquitin chains in the absence of an E3 (Hofmann and Pickart, 1999; McKenna et al., 2001; Wu et al., 2003; Andersen et al., 2005; Pastushok et al., 2005). By adapting methods used in these studies, the in vitro ubiquitin conjugation assays described here were established to delineate the ubiquitination activities of UBC2 and UEV1: firstly, to assess the cooperation of UBC2 and UEV1 in di-ubiquitin formation and, secondly, to assess the cooperation of UBC2 and UEV1 with E3 ubiquitin ligases (Figure 1). The method described here allows for the assessment of di-ubiquitin formation by an E2 and E2 variant pair and their collective cooperation with an E3 ligase. The assay uses western blotting specifically to detect ubiquitin conjugates. Blotting with an antibody specific to K63-conjugation also enables the assessment of whether the ubiquitin-conjugate products are linked at K63. Although we have not tested other antibodies, we anticipate that the method could easily be adapted to assay antibodies specific to other ubiquitin linkages.

Figure 1. Ubiquitination outcomes mediated by UBC2 and UEV1. In the presence of ATP, ubiquitin (Ub) is activated and covalently attached in step 1 to the E1 UBA1a. In the presence of UEV1 and UBC2, the ubiquitin is transferred in step 2 to the UBC2 component of the UBC2–UEV1 heterodimer, and subsequently in step 3 onto a second ubiquitin molecule to form a K63-linked ubiquitin dimer, as described in Assay A. In the absence of UEV1, a UBC2–Ub complex is formed in step 4, which in the presence of an E3 such as RNF8 or BIRC2 forms polyubiquitin chains that may be unanchored or anchored to an unidentified substrate protein as described in Assay B. The linkage(s) in these chains are not known but are not K63-based.