Figure 2.
Mutations which attenuate the scramblase activity of ATG-9 suppress the defect in epg-5 mutants. (A) The membrane topology of monomeric C. elegans ATG-9. Each monomer contains six membrane-penetrating domains. TM and RM represent the predicted transmembrane and reentrant membrane helices, respectively. The ATG-9(C475F) mutation in atg-9(bp1607), atg-9(bp1532), and atg-9(bp1860) is marked by a red asterisk. The ATG-9(G476W) mutation in atg-9(bp1719) is marked by a blue asterisk, the ATG-9(R483W) mutation in atg-9(bp1720) by a magenta asterisk, and the ATG-9(K382L R383L R384L) mutations in atg-9(bp1723) by a green asterisk. (B) Left: Superimposition of C. elegans ATG-9 (residues 102–582) with tertiary structures predicted by AlphaFold3. Right: Cryo-EM structure of human ATG9A (PDB ID: 7JLO) (Maeda et al., 2020). These structures reveal that the homotrimer forms a central pore. In the C. elegans ATG-9 structure on the left, amino acids marked in red, blue, and magenta represent C475, G476, and R483 in TM4, which alter the width and hydrophilicity of the central pore. In the human ATG9A structure on the right, green and cyan represent charged residues in RM1 (Lys321, Arg322, and Glu323) and small amino acids in TM4 (Thr410, Thr412, Gly415, and Thr419), respectively, which together form the wide, hydrophilic central region of the pore, which is crucial for lipid scramblase activity. (C–E) A large number of GFP::SEPA-1 aggregates accumulate in epg-5 mutant embryos at the comma stage (C′), and this accumulation is suppressed in epg-5; atg-9(bp1719) animals (D) and epg-5; atg-9(bp1723) animals (E). The dashed contours in D and E indicate the embryo outlines. C shows the DIC images of the embryos in C′. (F) Quantification of the number of GFP::SEPA-1 aggregates in embryos of the indicated genotypes (n = 3 embryos for each genotype). Data are shown as mean ± SEM; ***, P < 0.001. Scale bars: 5 μm for C–E.

Mutations which attenuate the scramblase activity of ATG-9 suppress the defect in epg-5 mutants. (A) The membrane topology of monomeric C. elegans ATG-9. Each monomer contains six membrane-penetrating domains. TM and RM represent the predicted transmembrane and reentrant membrane helices, respectively. The ATG-9(C475F) mutation in atg-9(bp1607), atg-9(bp1532), and atg-9(bp1860) is marked by a red asterisk. The ATG-9(G476W) mutation in atg-9(bp1719) is marked by a blue asterisk, the ATG-9(R483W) mutation in atg-9(bp1720) by a magenta asterisk, and the ATG-9(K382L R383L R384L) mutations in atg-9(bp1723) by a green asterisk. (B) Left: Superimposition of C. elegans ATG-9 (residues 102–582) with tertiary structures predicted by AlphaFold3. Right: Cryo-EM structure of human ATG9A (PDB ID: 7JLO) (Maeda et al., 2020). These structures reveal that the homotrimer forms a central pore. In the C. elegans ATG-9 structure on the left, amino acids marked in red, blue, and magenta represent C475, G476, and R483 in TM4, which alter the width and hydrophilicity of the central pore. In the human ATG9A structure on the right, green and cyan represent charged residues in RM1 (Lys321, Arg322, and Glu323) and small amino acids in TM4 (Thr410, Thr412, Gly415, and Thr419), respectively, which together form the wide, hydrophilic central region of the pore, which is crucial for lipid scramblase activity. (C–E) A large number of GFP::SEPA-1 aggregates accumulate in epg-5 mutant embryos at the comma stage (C′), and this accumulation is suppressed in epg-5; atg-9(bp1719) animals (D) and epg-5; atg-9(bp1723) animals (E). The dashed contours in D and E indicate the embryo outlines. C shows the DIC images of the embryos in C′. (F) Quantification of the number of GFP::SEPA-1 aggregates in embryos of the indicated genotypes (n = 3 embryos for each genotype). Data are shown as mean ± SEM; ***, P < 0.001. Scale bars: 5 μm for C–E.

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