Lysine residues 348 and 352 are important for the ubiquitination of VPS-34 in vitro and in vivo. (A and B) Mutations of lysine 348 (K348) and lysine 352 (K352) abrogated poly- (A) and mono (B)-ubiquitination of VPS-34 in vitro. FLAG IP was performed followed by detection of ubiquitination with anti-HA antibodies. (C) Ubiquitination of VPS-34 was examined in WT and vps-34(qx546) mutants carrying both ha::ubq-2 and flag::vps-34 (WT) or flag::vps-34 (K348RK352R) (vps-34[qx546]) generated by CRISPR-Cas9. The graph shows quantification of the level of ubiquitination using ImageJ (version 1.48v). The ratio of ubiquitin versus FLAG-VPS-34 was determined and normalized to onefold in WT. At least three independent experiments were performed. Data are shown as mean ± SD and were compared using the unpaired t test. **, P < 0.0001. (D) Cell corpses were scored at the twofold embryonic stage in the indicated strains. (E–G′) DIC and fluorescent images of cell corpses in piki-1(ok2346), vps-34(qx546), and vps-34;piki-1 embryos expressing YFP::2XFYVE. The head region of embryos at the twofold stage is shown. Cell corpses labeled by YFP::2XFYVE are indicated by white arrowheads whereas unlabeled corpses are indicated by yellow arrowheads. (H) The percentage of cell corpses labeled by YFP::2xFYVE in WT, vps-34(qx546), chn-1(by155), and vps-34(qx546);piki-1 was quantified. In (D and H), at least 15 embryos were scored in each strain and data are shown as mean ± SD. One-way ANOVA with Tukey’s posttest was performed to compare datasets linked by lines (D) or mutant datasets with WT (H). **, P < 0.0001; N.S., no significance. Bars, 5 µm.