Fig 2: IP3R-mediated elevation in Ca2+ levels is required for TBC1D9 recruitment to bacteria and TBK1 activation.a Representative trace of Fura 2-AM ratio during GAS WT or ?slo infection. b–d HeLa cells expressing EmGFP-LC3 and mRuby2-TBC1D9 and treated with or without BAPTA-AM (10 µM) were infected with GAS for 4 h. c Confocal images of TBC1D9 recruitment during infection and d quantification of cells harboring TBC1D9-positive bacteria, and d quantification of colocalization between LC3 and TBC1D9. e Effects of BAPTA-AM treatment on TBK1 phosphorylation during GAS infection. f Effects of IP3Rs depletion on TBK1 phosphorylation during GAS infection. g, h Recruitment of ULK1 to intracellular GAS in IP3Rs-KD cells. HeLa cells transfected with mClover-ULK1 and the indicated siRNAs were infected with GAS, fixed at 4 h, and stained for endogenous ubiquitin. g Representative confocal images and h the frequency of ULK1-positive bacteria. i, j HeLa cells transfected with EmGFP-LC3 and the indicated siRNAs were infected with GAS for 4 h. i Representative confocal images and j the frequency of cells with LC3-positive bacteria. k HeLa cells transfected with the indicated siRNAs were infected with GAS and intracellular bacterial CFU was determined at 6 h p.i. Data in (c) (n > 200 cells per condition), (d) (n > 5 images per condition), (h) (n > 50 bacteria per condition), (j) (n > 200 cells per condition), and (k) represent the mean ± SEM of three independent experiments. P values calculated by two-tailed Student’s t test. **P value < 0.01, ***P value < 0.001.

Fig 3: Model of Ca2+-dependent TBC1D9 and TBK1 activation in xenophagy and mitophagy.In xenophagy, TBC1D9 is activated by ER-derived Ca2+ and recruits to ubiquitin (Ub). Then recruited TBC1D9 activates TBK1 and activated TBK1 regulates NDP52 recruitment to Ub. Finally, ULK1 complex is recruited by NDP52/Ub/RAB35 for initiation of autophagy. In mitophagy, TBC1D9 targets depolarized mitochondria in a Parkin-dependent manner and facilitates TBK1 activation on the mitochondria. TBK1 mediates NDP52 recruitment for mitophagy.

Fig 4: TBC1D9 is involved in xenophagy during GAS infection.a, b Recruitment of the indicated proteins to invading GAS. a Representative confocal images and b quantification of GAS-positive cells. c, d Conversion of LC3-I to LC3-II. c Representative western blot of LC3 during starvation and GAS infection and d quantification of LC3-II/LC3-I ratio. e, f Localization of ULK1 in GAS-infected cells. e Representative confocal images of ULK1 recruitment to ubiquitin-coated GAS and f quantification of ULK1-positive GAS. g Intracellular bacterial CFU at 6 h p.i in WT and TBC1D9-KO HeLa cells. Scale bars, 10 µm. Data in (b) (n > 200 cells per condition), (d, f) (n > 50 bacteria per condition), and (g) represent the mean ± SEM of three independent experiments. P values calculated by two-tailed Student’s t test.

Fig 5: TBC1D9 recruitment to invading GAS requires its Ca2+-binding site and UBR.a, b TBC1D9 recruitment to GAS. HeLa cells expressing GFP-TBC1D9 and mCherry-LC3 were infected with GAS, fixed, and stained with DAPI. a Representative confocal images of GFP-TBC1D9 localization in uninfected or GAS-infected cells and b the time course of TBC1D9, ubiquitin, and LC3 recruitment during GAS infection. c HeLa cells were infected with GAS WT or the ?slo mutant for 4 h, fixed, and immunostained for TBC1D9. The percentage of TBC1D9-positive GAS-infected cells is shown. d WT or ATG5-KO cells expressing GFP-TBC1D9 were infected with for 4 h, and the percentage of TBC1D9-positive GAS-infected cells is shown. e Domain organization and deletion mutants of TBC1D9. f Localization of GFP-TBC1D9 deletion mutants during GAS infection. g Beads coated with GST, GST-ubiquitin, or ubiquitin-like proteins were incubated with lysate from cells expressing GFP-TBC1D9, followed by immunoblot with an antibody against GFP. h, i HeLa cells infected with GAS WT or the ?slo mutant were stained with TBC1D9 and primary antibodies against Lys63-specific ubiquitin to assess TBC1D9-ubiquitin binding by Duolink PLA. h Representative confocal micrographs and i quantification of the PLA signal per cell. Dots (red) indicate TBC1D9-ubiquitin complexes. Scale bars, 10 µm. j GST pulldown assay with indicated TBC1D9 construct and GST as a control. GST-fusion proteins were incubated with purchased diubiquitins linked by Lys48 or Lys63 (Boston Biochem) and analyzed by immunoblotting with antiubiquitin antibody. k, l HeLa cells expressing EmGFP-TBC1D9 constructs were infected with GAS, fixed, and stained with primary antibodies against anti-GFP and anti-Lys63-specific ubiquitin to assess TBC1D9-ubiquitin binding by Duolink PLA. k Representative confocal micrographs and l quantification of the PLA signal per cell. Data in (b) (n > 200 cells per condition), (c) (n > 200 cells), (d) (n > 200 cells), (i) (n > 20 cells per condition), and (l) (n > 20 cells per condition) represent the mean ± SEM of three independent experiments. P values calculated by two-tailed Student’s t test.