Figure 7. Increasing K6-linked Ub conjugates impairs parkin-mediated mitophagy.

A, B Expression of HA-Ub^{K6 only} delays the mitochondrial recruitment of parkin following treatment with CCCP. U2OS-GFP-parkin cells were transfected with either HA-Ub^wild-type, HA-Ub^{K6 only}, or HA-Ub^K6R (2 μg) for 60 h (A). Cells were treated with CCCP for indicated time periods and fixed. Immunofluorescence images of cells were acquired after staining for HA and TOM20. After 1-h treatment with CCCP, cells overexpressing either HA-Ub^wild-type, HA-Ub^{K6 only}, or HA-Ub^K6R (2 μg) were analyzed for the recruitment of GFP-parkin onto TOM20-positive mitochondria in HA-positive cells (B). Experiments were blinded and performed in triplicate with 100 cells analyzed for each condition. The vertical bars represent SEM. For statistical analysis, a two-way ANOVA with Tukey post-test was performed, **P < 0.01.

C, D Expression of HA-Ub^{K6 only} impairs parkin-mediated mitophagy. U2OS-GFP-parkin cells were transfected with either HA-Ub^wild-type, HA-Ub^{K6 only}, or HA-Ub^K6R (2 μg) for 60 h (C). Cells were treated with CCCP for 24 h and fixed. Immunofluorescence images of cells were acquired after staining for HA and TOM20. After 24 h treatment with CCCP, the percentage of HA-positive cells negative for TOM20 staining was quantified (D). Experiments were blinded and performed in triplicate with 100 cells analyzed for each condition. For statistical analysis, a two-way ANOVA with Tukey post-test was performed, **P < 0.01.

E Schematic for how USP8 knockdown delays parkin-mediated mitophagy. At steady state, parkin is auto-inhibited with minimal levels of auto-ubiquitination observed. Ub chains that do form can be removed by USP8 acting upon the K6 linkages within these conjugates. However, when USP8 is absent, Ub conjugates persist on parkin, whose presence appears to delay parkin recruitment onto depolarized mitochondria by impeding the interaction of parkin with either PINK1/phosphorylated Ub or another parkin activating protein. During mitophagy, a robust increase in parkin auto-ubiquitination occurs, followed by removal of Ub conjugates and subsequent clearance of damaged mitochondria. When USP8 is knocked down, these chains are now left intact. Thus, their presence delays parkin-mediated clearance by potentially impeding parkin from interacting with p62, LC3, or another autophagic adaptor protein required for the successful completion of mitophagy.