Figure 5. Self-oligomerization of p62 is... | Rockefeller University Press

Figure 5.

$Figure 5. Self-oligomerization of p62 is critical for its localization to the autophagosome formation site. (A) Schematic representation of the artificial p62 oligomerization system using a FKBP domain. The PB1 domain of p62 was replaced by one FKBP or two tandem FKBP domains. The small ligand AP20187 chemically links two FKBP domains. (B) p62 KO MEFs stably expressing GFP-1xFKBP-p62 or GFP-2xFKBP-p62 were treated with ethanol (vehicle) or 0.1 µM AP20187 for 24 h. After homogenization and centrifugation, the resulting supernatant fractions were applied to a Superose 6 column. Each fraction was analyzed by immunoblotting using anti-p62 antibodies. Positions of the molecular mass standards (kD) are shown. (C–E) p62 KO MEFs stably expressing HA-ULK1 and either GFP-1xFKBP-p62 or GFP-2xFKBP-p62 were treated with ethanol (vehicle) or 0.1 µM AP20187 for 24 h and cultured in regular or starvation medium containing 0.2 µM wortmannin with or without 0.1 µM AP20187 for 1 h. Cells were analyzed by immunofluorescence microscopy using anti-HA antibodies. The number of p62 puncta per cell (D) and the ULK1 positivity (%) of the p62 puncta (E) are shown. Data represent mean ± SEM of 30 images. Signal color is indicated by color of typeface. AP, AP20187; WM, wortmannin. (F) Proposed model of p62 localization to autophagic structures. Bars: (white) 10 µm; (yellow) 1 µm.$

Self-oligomerization of p62 is critical for its localization to the autophagosome formation site. (A) Schematic representation of the artificial p62 oligomerization system using a FKBP domain. The PB1 domain of p62 was replaced by one FKBP or two tandem FKBP domains. The small ligand AP20187 chemically links two FKBP domains. (B) p62 KO MEFs stably expressing GFP-1xFKBP-p62 or GFP-2xFKBP-p62 were treated with ethanol (vehicle) or 0.1 µM AP20187 for 24 h. After homogenization and centrifugation, the resulting supernatant fractions were applied to a Superose 6 column. Each fraction was analyzed by immunoblotting using anti-p62 antibodies. Positions of the molecular mass standards (kD) are shown. (C–E) p62 KO MEFs stably expressing HA-ULK1 and either GFP-1xFKBP-p62 or GFP-2xFKBP-p62 were treated with ethanol (vehicle) or 0.1 µM AP20187 for 24 h and cultured in regular or starvation medium containing 0.2 µM wortmannin with or without 0.1 µM AP20187 for 1 h. Cells were analyzed by immunofluorescence microscopy using anti-HA antibodies. The number of p62 puncta per cell (D) and the ULK1 positivity (%) of the p62 puncta (E) are shown. Data represent mean ± SEM of 30 images. Signal color is indicated by color of typeface. AP, AP20187; WM, wortmannin. (F) Proposed model of p62 localization to autophagic structures. Bars: (white) 10 µm; (yellow) 1 µm.