Golgi membrane-associated degradation pathway in yeast and mammals (2016)
Figure 4

Figure 4. Pharmacological and genetic induction of GOMED in Atg5-deficient MEFs

(A−I) Induction of GOMED in Atg5 KO MEFs by CBM treatment. (A, B) EM analysis revealed the formation of AP-like and AL-like structures. Atg5 KO MEFs were treated with CBM (2 mM) for 3 hr. A representative low-magnification image (A) and high-magnification images (B) are shown. Scale bar = 1 µm (A) and 0.2 µm (B). The arrowheads and arrows indicate AL-like and AP-like structures, respectively. (C-F) Involvement of Golgi membranes in the generation of AL-like structures. (C) CLEM analysis of mCherry–syntaxin 6-expressing Atg5 KO MEFs. Cells were treated with CBM (2 mM) for 24 hr and observed using fluorescence and electron microscopy. mCherry–syntaxin 6 puncta merged with the AL-like structures. Scale bar = 5 µm. “N” indicates nucleus. A magnified image of the dashed square is shown in the inset. Scale bars = 2 µm. (D, E) Requirement of Grasp65 in the generation of AL-like structures. Atg5 KO and Grasp65-silenced Atg5 KO MEFs were treated without (NT) or with CBM (2 mM) for the indicated times followed by immunostaining with an anti-Lamp2 antibody. Representative images (at 24 h) are shown in (D). Scale bars = 5 µm. A magnified image of the dashed square is shown in the inset. Scale bars = 1 µm. CBM induced large ring-like Lamp2 fluorescence. (E) Percentages of cells with large ring-like Lamp2 immunofluorescence (mean ± s.e.m., n = 4). *p < 0.01 vs the value of Atg5 KO MEF. (F) Colocalization of Lamp2 and GFP–syntaxin 6 in CBM-treated Atg5 KO MEFs. GFP–syntaxin 6-expressing Atg5 KO MEFs were treated with (CBM) or without (NT) CBM (2 mM) for 6 hr and immunostained with an anti-Lamp2 antibody. The ring-like Lamp2 fluorescence merged with the signal for syntaxin 6. Scale bar = 5 µm. A magnified image of the dashed square is shown in the inset. Scale bars = 2 µm. (G) The monomeric red fluorescent protein (mRFP)–green fluorescent protein (GFP) tandem protein assay revealed the induction of GOMED. Atg5 KO MEFs stably expressing tandem mRFP–GFP were incubated without (no treatment) or with CBM (2 mM) for 24 hr. Red signals indicate acidic compartments. Lysosomes were counterstained with an anti-Lamp2 antibody (cyan). Scale bars = 5 µm. Regions of interest (ROI) are indicated by dashed squares. Arrowheads indicate GOMED structure. Scale bars = 2 µm. (H, I) Degradation of GFP-fused proteins indicated the induction of GOMED. Atg5 KO MEFs stably expressing VSVG–GFP (H) or M6PR–GFP (I) were incubated with CBM (5 mM) for the indicated times. Cell lysates were subjected to immunoblotting with the anti-GFP antibody. (J−M) Genetic induction of GOMED in Atg5 KO MEFs. Atg5 KO MEFs were treated with the indicated small interfering (si)RNAs for 24 hr and GOMED was assessed by EM (J-L) and the mRFP–GFP tandem protein assay (M). Representative images are shown in (J, K). Arrows indicate AL-like structures. Scale bars = 2 µm. (L) The number of AP-like and AL-like structures was calculated from the EM images of cells treated with the indicated siRNAs. Error bars indicate s.e.m. (siControl: n = 23, siPI4k2α + 3β: n = 20, siArfaptin1: n = 20). *p < 0.01 vs the value of siControl. (M) The same experiments as in (G) were performed using cells treated with the indicated siRNAs. Representative images are shown. Arrowheads indicate GOMED structure. Scale bars = 5 µm.