Hcy binding and homocysteinylation interfere with VHA-12 function. (A) V-ATPase activity is markedly decreased in cbs-1 mutants. VHA-12::GFP was immunoprecipitated from yqSi11 (single-copy insertion of VHA-12::GFP) and yqSi11;cbs-1(yq357) animals and detected with GFP antibody. Coprecipitated endogenous VHA-13 and VHA-8 were detected with VHA-13 (top left) and VHA-8 (bottom left) antibodies. Lysates from WT and cbs-1(yq357) animals without expression of VHA-12::GFP were used as control (Ctrl) in both IP experiments and V-ATPase assays. The precipitated VHA-12::GFP-containing complex was subject to V-ATPase activity assays at the indicated reaction time points (right). Data (mean ± SEM) are from three independent experiments. (B) Reinforced VHA-12::GFP expression ameliorates the defective lysosomes in cbs-1(yq357) mutants. Representative images of NUC-1::mCh-labeled lysosomes in WT, cbs-1(yq357) (non-transgenic) mutants, and cbs-1(yq357) mutants with reinforced expression of VHA-12::GFP (P_vha-12vha-12::gfp). Bars, 5 μm. ≥20 lysosomes from 3 animals (2 days after L4) were examined for each genotype and data (mean ± SEM) are from three independent experiments. (C) VHA-12::GFP expression rescues the body length defect of cbs-1(yq357) mutants. Left: Representative DIC images of adults (2 days after L4). Bars, 200 μm, and right: Quantification of body length (mean ± SEM) from 3 independent experiments, ≥15 animals per genotype. (D) GFP::VHA-13 overexpression fails to suppress lysosomal defects in cbs-1(yq357) mutants. Representative images and quantification of NUC-1::mCh distribution in WT and cbs-1(yq357) backgrounds expressing GFP::VHA-13. Analysis performed as described in B. (E) Representative images (left) and quantification (right) of lysosomes (NUC-1::mCh) in WT and cbs-1(yq357) animals overexpressing VHA-12(K90A)::GFP, VHA-12(C269A)::GFP, and VHA-12(C269Q)::GFP. Bars, 5 μm. ≥20 lysosomes from 3 animals (2 days after L4) were examined for each genotype, and data (mean ± SEM) are from three independent experiments. For all quantifications, statistical significance was calculated using the two-sided Student’s t test. DIC, differential interference contrast. ***P < 0.001 and ****P < 0.0001. Source data are available for this figure: SourceData F6.