Fig. 7: SPATA33 interaction with ATG16L1 and VDAC2.

a, b Coimmunoprecipitation between VDAC2 and deletion mutants of SPATA33. pCherry-FLAG-ATG16L1 was transiently co-transfected with pGFP-SPATA33, pGFP-N-SPATA33, or pGFP-C-SPATA33 in HEK293T cells, respectively. Cell lysates were examined by western blotting using the anti-FLAG or anti-GFP antibody. For coimmunoprecipitation, the lysates were immunoprecipitated with anti-FLAG or anti-GFP, followed by immunoblotting with the anti-GFP or anti-FLAG antibody. Arrowheads indicate the target bands. c, d Coimmunoprecipitation between ATG16L1 and deletion mutants of SPATA33. p3xFLAG-VDAC2 was transiently co-transfected with pGFP-SPATA33, pGFP-N-SPATA33, or pGFP-C-SPATA33 in HEK293T cells, respectively. Cell lysates were examined by western blotting using the anti-FLAG or anti-GFP antibody. For coimmunoprecipitation, the lysates were immunoprecipitated with anti-FLAG or anti-GFP, followed by immunoblotting with the anti-GFP or anti-FLAG antibody. Arrowheads indicate the target bands. e Coimmunoprecipitation analysis of interaction among endogenous SPATA33, ATG16L1, and VDAC2 in GC-1 cells. The GC-1 cell lysates were immunoprecipitated with anti-SPATA33, anti-ATG16L1, or anti-VDAC2 antibody, followed by immunoblotting with the anti-SPATA33, anti-VDAC2, or anti-ATG16L1 antibody, respectively. The whole cell lysates were examined by western blotting using the anti-ATG16L1, anti-VDAC2, or anti-SPATA33 antibody. Arrowheads indicate the target bands. f Colocalization analysis of SPATA33 with VDAC2 and ATG16L1. GC-1 cells were transiently co-transfected with pCherry-SPATA33 and pGFP-VDAC2. After 24 h in normal medium, the cells were cultured in normal (control), CCCP (10 μM, 1 h), EBSS medium (1 h), or EBSS with CCCP (10 μM, 1 h) addition, respectively. Immunofluorescence analysis was performed with anti-ATG16L1 and Dylight 405 Donkey anti-Rabbit IgG (H + L) antibodies. Single channel (red, green, or blue) and merged images were taken by confocal microscopy. Colocalizing structures are indicated in white (merge). Scale bar: 25 μm. g Statistical analysis of colocalized puncta between SPATA33, ATG16L1, and VDAC2. Data are presented as means ± S.D. **p < 0.01 (n = 3 independent experiments, >15 cells per experiment). h SPATA33 mediates mitophagy via interaction with VDAC2 and ATG16L1. SPATA33 interacts with mitochondrial outer membrane protein VDAC2 through its carboxyl terminal, while its amino terminal interacts with WD40 region of ATG16L1 protein. Upon starvation stress, damaged mitochondria are encapsulated by autophagosome through mediator SPATA33 linking between VDAC2 and ATG16L1 to initiate mitophagy.