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. 2010 Nov 16;6(8):1090–1106. doi: 10.4161/auto.6.8.13426

Figure 7.

Figure 7

Anchoring ubiquitin to outer mitochondrial membrane is sufficient for p62 recruitment and mitochondrial clustering but not mitophagy. (A) HeLa cells, which lack Parkin protein expression, were co-transfected with ubiquitin G76V fused to tandem FKBP12 domains and mitochondrial anchor constructs fused to the FRB domain. The cells were subsequently treated with the rapalog AP21967 for 24 hrs. Rapalog induced the translocation of ubiquitin to mitochondria secondary to heterodimerization of the FKBP and FRB domains in the presence of the rapalog. Cells were immunostained with Tom20 and the cells were scored for the absence of mitochondria. ≥50 cells per condition were counted. (B) HeLa cells co-transfected with EGFP-Ubiquitin G76V-FKBP or FKBP-EGFP and FRB-Fis1 (92–152) were treated with rapalog and CCCP for 24 hrs and immunostained for p62 (red) and the mitochondrial protein Tom20 (white). Scale bar in last image = 10 µm. (C) Cells treated as in (B) were scored for the presence of p62 positive and p62 negative mitochondrial clumps. ≥50 cells per condition were counted.