FIGURE 3:

Gp78 ubiquitin ligase activity induces mitophagy of depolarized mitochondria. (A) Cos7 cells were transfected with Flag-gp78 or Flag-RINGmut or cotransfected with Flag-gp78 and either HA-Ub or HA-Ubmono, as indicated, and (B) HEK293 cells were transfected with Flag-gp78 or Flag-RINGmut. Cells were either untreated (CTL) or treated with CCCP for 24 h and labeled with anti-Flag and mitochondrial OxPhosV Abs. Bar graphs show the percentage loss of mitochondrial mass of CCCP-treated cells relative to untreated cells ((mitochondrial area[CTL − CCCP]/mitochondrial area[CTL]) ×100). For both A and B, mean ± SEM; 10–20 cells/experiment; n = 3, **p < 0.01; see Supplemental Table S1 for mitochondrial area values. (C) HEK293 cells were transfected for 24 h with either control siRNA or ATG5-specific siRNA and then transfected with Flag-gp78 for a further 24 h. Cell lysates were blotted with antibodies to Mfn1, Mfn2, OxPhosV, ATG5, and β-actin, and anti-Flag immunoprecipitates were probed with anti-Flag antibodies to reveal presence of Flag-Gp78.