Fig 2: Transcription factor EB (TFEB) and Rab7 are involved in syntaxin 17 (STX17)-initiated mitophagy upon mitochondrial fission 1 protein (Fis1) loss. a Wild-type (WT) or Fis1 knockout (KO) HeLa cells were co-transfected with plasmids encoding green fluorescent protein (GFP)-Rab7 (green) and blue fluorescent protein (BFP)-STX17 (blue) for 24 h. Cells were fixed and immunostained for Tim23 (red) and Lamp2 (cyan). Cells were visualized as three-dimensional reconstruction images. b Fis1 KO HeLa cells were transfected with control-, TBC1D15-, or Rab7-small interfering RNA (siRNA) for 24 h. Cells were transiently transfected with GFP-tagged vector or STX17 (green) for further 24 h. Mitochondria and autophagosome were highlighted by Tim23 (red) and LC3 (cyan). Hoechst, blue. Scale bar, 10 µm. Arrowhead indicates mitophagic cell. c Quantification of mitophagic cells in b. ***P < 0.001 (two-tailed unpaired Student’s t test from three independent experiments, n = 150). d Fis1 KO cells were transfected with control- or Rab7-siRNA for 24 h. Cells were then again transfected with respective siRNA, plus GFP-tagged vector or STX17 for further 6 h. Hereafter, cells were cultured with or without chloroquine (CQ) for another 66 h. Cells were harvested and lysed, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel separation and immunoblotting against overall mitochondrial proteins. e WT or Fis1 KO HeLa cells were co-transfected with GFP-tagged vector or STX17 (green) and TFEB-Flag (cyan) for 24 h. Cells were fixed and immunostained against Tom20 (red) and Flag (cyan). Hoechst, blue. Scale bar, 10 µm. Arrowhead indicates mitophagic cell. f WT or Fis1 KO HeLa cells treated as in e were fractioned, lysed, and immunoblotted. Asterisk indicates non-specific bands. g Quantification of f. TFEB-Flag expression level was normalized to Tubulin (cytosol) or C23 (nuclear). Nuclear TFEB-Flag was indicated as the percentage of total TFEB-Flag. **P < 0.01, NS, not significant (two-tailed unpaired Student’s t test, n = 3). Error bars represent SD (c and g)

Fig 3: Mitochondrial fission 1 protein (Fis1) depletion accompanies mitochondrial respiration dysfunction. a Oxygen consumption rate (OCR) was measured in wild-type (WT) and Fis1 knockout (KO) HeLa cells. Dashed vertical lines indicate the addition of 1 µM oligomycin (Olig), 0.5 µM carbonyl cyanide 3-chlorophenylhydrazone (CCCP), and 1 µM rotenone plus 1 µM antimycin A (Rot/Ant), respectively (n = 8). b Individual parameters for basal respiration and maximal respiration analysis of a. **P < 0.01, *P < 0.05 (two-tailed unpaired Student’s t test, n = 3). c Glucose or galactose-cultured Fis1 KO HeLa cells were transiently transfected with green fluorescent protein (GFP)-tagged vector or syntaxin 17 (STX17) for 24 h. Cells were fixed and immunostained with anti-Tim23 (red) and anti-LC3 (cyan) antibodies. Hoechst, blue. Scale bar, 10 µm. d Quantification of c. ***P < 0.001 (two-tailed unpaired Student’s t test). n = 150 GFP-positive cells/group from three replicates. e Plasmids as indicated were transfected into Fis1 KO HeLa cells. At 6 h post-transfection, cells were cultured in medium with glucose or galactose for further 18 h. Cells were extracted and co-immunoprecipitated with anti-Flag beads, followed by immunoblotting with respective antibodies. The fold change of the interaction between ATG14 and STX17 is shown as mean ± SD from three independent experiments. f GFP-tagged STX17 was transfected into Fis1 KO HeLa cells for 6 h. Then, cells were cultured in medium with glucose or galactose for further 18 h. Immunoblotting of subcellular fractions were analyzed. PNS, post-nuclear supernatant; CYTO, cytosol; ER, endoplasmic reticulum; MAM, mitochondria-associated membranes; MITO, mitochondria. g, h Quantification of relative ratio of GFP-tagged STX17 (g) or endogenous ATG14 (h) in the respective subcellular fractions. *P < 0.05 (two-tailed unpaired Student’s t test, n = 3). Error bars represent SD (a, b, g and h)

Fig 4: Syntaxin 17 (STX17)-induced mitophagy is PINK/Parkin-independent. a Wild-type (WT) or mitochondrial fission 1 protein knockout (Fis1 KO) HeLa cells were transfected with Flag-tagged vector or STX17 and green fluorescent protein (GFP)-tagged Parkin (green) for 24 h. Cells were analyzed by immunofluorescence microscopy using antibodies against Tom20 (red) and Flag (cyan). Hoechst, blue. Scale bar, 10 µm. White arrow indicates cell with STX17 overexpression-mediated mitophagy. b Bar chart shows the percentage of cells with Parkin translocation as indicated in a. Parkin translocation upon carbonyl cyanide 3-chlorophenylhydrazone (CCCP) treatment at 10 µM for 4 h was quantified as positive control. Data are mean ± SD (n = 150 cells from three independent experiments). ***P < 0.001 (two-tailed unpaired Student’s t test). c WT or Fis1 KO HeLa cells were incubated with dimethyl sulfoxide (DMSO) or 10 µM CCCP for 16 h and then fixed and immunostained for Tim23 (red) and Fis1 (green). Cells transiently expressing GFP-tagged Parkin and treated with 10 µM CCCP for 16 h were used as a positive control for mitophagy. Hoechst, blue. Scale bar, 10 µm. d HeLa cells expressing GFP-tagged vector or STX17 were treated with 10 µM CCCP for 16 h and immunostained for Tim23 (red). Hoechst, blue. Scale bar, 10 µm. e WT or Fis1 KO HeLa cells were transfected with GFP-tagged vector or STX17 for 72 h. SH-SY5Y cell lysate was used as the positive control for the expression of endogenous Parkin. Cell lysates were immunoblotted for Parkin and mitochondrial proteins. f HeLa cells stably expressing GFP-Parkin (green) were transfected with small interfering RNA (siRNA) against control, Fis1 or STX17 for 48 h. Then, cells were treated with or without 10 µM CCCP for further 4 h. Cells were visualized by immunostaining for Tim23 (red) and LC3 (cyan). Hoechst, blue. Scale bar, 10 µm

Fig 5: Syntaxin 17 (STX17) initiates mitophagy upon mitochondrial fission 1 protein (Fis1) depletion. a HeLa cells were transfected with respective small interfering RNAs (siRNAs) for 24 h, before transient transfection with green fluorescent protein (GFP)-tagged vector or STX17 (green) for further 24 h. Cells were fixed and immunostained with Tim23 (red) and LC3 (cyan) antibodies. Hoechst, blue. Scale bar, 10 µm. White arrowhead indicates mitophagic cell. b Quantification of cells with decreased mitochondria as shown in a. Data are mean ± SD (n = 150 cells from three independent experiments). ***P < 0.001 (two-tailed unpaired Student’s t test). c Fis1 knockout (KO) HeLa cells were transfected with GFP-tagged STX17 for 24 h. Cells were fixed and analyzed by immunofluorescence against Tim23 (red) and LC3 or P62 (cyan). Z-stack images were collected and a representative three-dimensional reconstruction example is shown. Hoechst, blue. Scale bar, 10 µm. d Wild-type (WT) or Fis1 KO HeLa cells were transiently transfected with GFP-tagged STX17 (green) for 24 h. Images were acquired by super-resolution structured illumination microscopy (SR-SIM) after staining for Tom20 (red) and Lamp2 (gray). Hoechst, blue. Scale bar, 10 µm. Enlarged image represents in three-dimensional reconstruction. White arrow indicates the signal of GFP-STX17. e WT or Fis1 KO HeLa cells were transiently transfected with GFP-tagged vector or STX17 for 6 h. Cells were cultured with or without chloroquine (CQ) for further 66 h. Cell lysates were immunoblotted for outer mitochondrial membrane (OMM), intermembrane space (IMS), inner membrane mitochondrial (IMM), and matrix proteins. f WT or Fis1 KO HeLa cells transiently expressing GFP-tagged STX17 were analyzed by conventional transmission electron microscopy. Scale bars, 0.2 µm. Yellow arrows indicate autophagic structures enclosing mitochondria. g WT or Fis1 KO HeLa cells stably expressing mitochondrial-targeted form of the fluorescent reporter Keima (mt-Keima) were transiently transfected with blue fluorescent protein (BFP)-tagged STX17 (blue). Live cells were visualized by confocal microscopy excited with 488 nm (green) and 543 nm (red). Scale bar, 10 µm. h WT or Fis1 KO HeLa cells stably expressing mt-Keima were transiently transfected with GFP-tagged vector or STX17 for 48 h. mt-Keima fluorescence in GFP-positive cells was measured using flow cytometry by excitation at 488 nm (neutral) and 543 nm (acidic). Data are mean ± SD (n = 3)