Figure 1
From: K63-linked ubiquitylation induces global sequestration of mitochondria
ProxE3: an engineered ubiquitin ligase for inducible K63-linked polyubiquitylation. (A) Schematic illustration of the ProxE3 ligase and its substrate GFP-Sub. Dimerization of the DmrC domain in ProxE3 and the DmrA domain in GFP-Sub is induced by the heterodimerizer AP21967 and results in specific K63-linked ubiquitylation of GFP-Sub. (B) In vitro heterodimerization of GFP-Sub and mCherry-ProxE3. Recombinant GFP-Sub was immobilized on GFP-Trap magnetic beads prior to addition of mCherry-ProxE3 in the presence or absence of AP21967. (C) One-hour in vitro ubiquitylation reaction using mCherry-ProxE3 or the catalytically inactive mCherry-ProxE3* in the presence or absence of AP21967, with or without immobilized GST-tagged GFP-Sub. (D) In vitro ubiquitylation kinetics of GFP-Sub by mCherry-ProxE3 or the catalytically inactive mCherry-ProxE3* and auto-ubiquitylation of the ligases. (E–G) One-hour in vitro ubiquitylation reaction of GFP-Sub by mCherry-ProxE3 with WT ubiquitin (WT Ub), a lysine-less ubiquitin mutant(UbK0) or single lysine ubiquitin mutants. Arrows are pointing towards the unmodified protein of interest (either GFP-Sub or ProxE3). (E) GFP detection, Western blot. (F) mCherry detection, Western blot. (G) Ubiquitin detection, Western blot.
