Figure 5. Phosphorylation of the polyUb chain activates Parkin E3 activity.

(A) The Ub SA mutation attenuates the CCCP-dependent Parkin-polyUb association, whereas the Ub SE mutation promotes this association. HEK293T cells were transfected with the indicated combination of plasmids and were then treated with 10 µM CCCP for 1 hr. Tom70^MTS-2FLAG-4Ub constructs (WT, SA and SE) were immunoprecipitated with anti-FLAG beads and eluted with 100 µg/ml 3xFLAG peptide. Coprecipitated GFP-Parkin was detected by western blot. Note that the expression of Tom70^MTS-2FLAG-4Ub SE alone did not lead to PINK1 accumulation (lane 7). The dot indicates putative ubiquitinated Mfn1. (B) Parkin associates with Tom70^MTS-2FLAG-4Ub SE through its RING1-IBR. HEK293T cells were transfected with a series of 3xMyc-tagged full-length Parkin and its mutants together with Tom70^MTS-2FLAG-4Ub SE. Coprecipitated Tom70^MTS-2FLAG-4Ub SE was detected with an anti-FLAG antibody. Diagram of the Parkin deletion mutants used in this experiment is shown at the bottom. (C) Phosphomimetic Parkin SE is activated by either cytosolic SE Ub or the mitochondrial SE polyUb chain. Note that the accumulation of PINK1 and obvious degradation of Mfn1 were not observed, even with a combination of HA-Ub SE and Tom70^MTS-2FLAG-4xUb SE (lane 12 vs. lane 1). The dot and the asterisk indicate putative ubiquitinated Mfn1 and non-specific signals, respectively.