Fig. 5.

Mitochondrial fission 1 protein (Fis1) regulates the translocation of syntaxin 17 (STX17) onto MAM/mitochondria. a, b Fis1 knockout (KO) HeLa cells were transfected with the indicated plasmids. After 24 h, cells were lysed and immunoprecipitated with anti-Flag beads followed by immunoblotting. Asterisk indicates a non-specific band. c Immunoblotting of subcellular fractions from wild-type (WT) or Fis1 KO HeLa cells expressing green fluorescent protein (GFP)-tagged STX17 for 24 h is shown. PNS, post-nuclear supernatant; CYTO, cytosol; ER, endoplasmic reticulum; MAM, mitochondria-associated membranes; MITO, mitochondria. d, e Quantification of relative ratio of GFP-tagged STX17 (d) or endogenous ATG14 (e) in the respective subcellular fractions. *P < 0.05, **P < 0.01 (two-tailed unpaired Student’s t test from three independent experiments). f WT or Fis1 KO HeLa cells were transfected with GFP-tagged STX17 (green). Cells were imaged following immunostaining for Tom20 (red) and FACL4 (fatty acid-CoA ligase 4) (cyan). Images at single focal plane were acquired. Hoechst, blue. Scale bar, 10 µm. Cropped image was enlarged and shown as a two-dimensional deconvoluted example. White arrows indicate the colocalization of STX17 with Tom20 (mitochondria) and FACL4 (MAM). g Line scans corresponding to f. The colocalization between GFP-STX17 with FACL4 (MAM) or Tom20 (mitochondria) of Fis1 KO cells is indicated by black triangles or purple triangles, respectively. h Plasmids encoding respective proteins were transfected into WT or Fis1 KO HeLa cells for 24 h. Cells were extracted and immunoprecipitated with anti-Flag beads, followed by immunoblotting. i Plasmids as indicated were transfected into HeLa cells. After 24 h, cells were extracted and co-immunoprecipitated with anti-Flag beads, followed by immunoblotting with anti-Myc and anti-Flag antibodies, respectively. j Fis1 KO HeLa cells were transfected with GFP-tagged plasmids (green) as indicated. Cells were fixed and stained with Tim23 (red) and LC3 (cyan) antibodies. Representative confocal microscopy images are shown. Hoechst, blue. Scale bar, 10 µm. Arrowheads indicate mitophagic cells. k Mitophagic cells as shown in j of Fis1 KO cells were quantified. ***P < 0.001, NS, not significant (two-tailed unpaired Student’s t test, n = 150 cells from three independent replicates). Error bars indicate SD (d, e, k)