Fig. 2.

TaiP targets ATG16L1’s WD40 domain. (A) Schematic of the ATG16L1 structure including the coiled-coil (C.C.) domain and the WD40 domain (WD40). The binding sites to ATG5, WIPI2B, and FIP200 are highlighted, along with the serine phosphorylated by ULK1. WD40 binding partners are indicated on the right. (B) WT or atg16l1 KO HeLa cells seeded on coverslips were transfected with the indicated constructs for 24 h. Cells were then infected with Ctr^ΔtaiP for 20 h at MOI = 0.2 before being fixed, permeabilized, and the inclusion membrane was stained with antibodies against the inclusion protein Cap1. The graph displays the median ± SD of three independent experiments (n > 50 cells in total) and the P values of the Student’s t test. (C) WT and HeLa cells were infected with either Ctr^WT or Ctr^ΔtaiP for 20 h before fixation, permeabilization, and immunostaining with antibodies against LC3B and the bacterial inclusion protein Cap1. LC3B is localized at the inclusion membrane independent of TaiP or ATG16L1. (Scale bar: 10 µm.) The dot plot displays the median intensity of LC3B staining at the inclusion membrane (n > 10 cells from three independent experiments). Student’s t tests showed no significant difference. (D) Lysates from atg16l1 KO cells transfected with either GFP-ATG16L1 or GFP-ATG16L1^1–319 were incubated with 100 pmol of recombinant GST-TaiP for 1 h at 4 °C, before performing a GST-PD using glutathione beads. Fractions were analyzed, such as in Fig. 1A. ATG16L1 was no longer pulled down withTaiP when its WD40 domain was deleted. (E) atg16l1 KO HeLa cells were cotransfected with Flag-TaiP and either GFP-ATG16L1 or GFP-ATG16L1^1–319. The cells were fixed 24 h later, permeabilized, and stained with anti-Flag antibodies. In the absence of the WD40 domain, Flag-TaiP was no longer recruited to ATG16L1 puncta. (Scale bar: 10 µm.)