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. Author manuscript; available in PMC: 2021 Jun 7.
Published in final edited form as: Nat Chem Biol. 2020 Dec 7;17(3):272–279. doi: 10.1038/s41589-020-00696-0

Figure 3 |. K63 chain forming HECT E3 ligases show strong preferences for a native lysine acceptor on ubiquitin.

Figure 3 |

(a) Cartoon of experimental scheme (left), monitoring reactivity of the yeast HECT E3 Rsp5p (middle) or human HECT E3 NEDD4 (right) and formation of di-UB chains with K63UBC1-C5 acceptors (UBA). (b) same as (a), except with K48UBC1-C5 acceptors. (c) HECT E3 ligase-dependent di-UB forming activity in the context of an Rsp5p-bound substrate (sortase-mediated UB, UB K63R, or K63UBC5 linkage to the WW-domain-binding PPPY degron motif of the substrate Sna4p. For all plots, di-UB levels (μMol) represent the final time-points from the reactions (Source Data Fig.3), N=2 independent experiments. For samples derived from the same experiment, gels were processed in parallel.