Figure 4.

ER-phagy requires a functional ATL GTPase domain and proper ER localization. (A) Schematic of the topology and dimerization of ATLs to facilitate ER membrane fusion. The different truncations and mutations of ATL2 tested are shown underneath. (B) Functional characterization of ATL2 domains required for ER morphology maintenance. U2-OS cells expressing mCherry-RAMP4 were transiently transfected with the indicated ATL2 constructs. Cells were then fixed and immunostained for HA-epitope. Scale bar represents 40 µm. Inset represents 3× enlargement of the boxed areas. (C) EATR CRISPRi HCT116 cells stably expressing sgATL2 and cDNA of ATL2 mutant variants were starved and analyzed by flow cytometry. Data presented as mean ± SD of three biological replicates. P value indicates one-way ANOVA with Dunnett’s multiple comparisons test (*, P < 0.05; ****, P < 0.0001). (D) CCER CRISPRi HCT116 cells with sgATL2 and stably expressing the indicated ATL2 mutants were starved, harvested, and subjected to Western blot. (E) Quantification of data from D. Data presented as mean ± SD of three biological replicates. P value indicates one-way ANOVA with Dunnett’s multiple comparisons test (*, P < 0.05).