V-ATPase is homocysteinylated in cbs-1 mutants. (A) Images of lysosomes (left) labeled with mK2::GFP::LGG-2 fusion protein in the hypodermis (Hyp) and muscle (Mus) of WT animals treated with control RNAi (Ctrl) or RNAi against vha-12 and vha-13. RNAi was performed RNAi-competent OP50 E. coli. Bars, 5 μm. Quantifications of the fluorescence intensity ratio (calculated as GFP/[GFP+mKate2]) are shown on the right. ≥20 lysosomes from 3 animals were analyzed for each group. Data (mean ± SEM) are from three independent experiments. (B) Hcy binds to VHA-12. Left: Coomassie blue staining of purified His₆-VHA-12. Right: binding of Hcy to His₆-VHA-12 in MST assays. Data represent the mean of three independent experiments. (C) Schematic illustration of protein modification by Hcy and HTL (Hcy thiolactone). The top row shows S-homocysteinylation of protein cysteine residues by Hcy, and the bottom row shows N-homocysteinylation of protein lysine residues by HTL. Filled orange ovals represent proteins. (D) Summary of homocysteinylation sites in VHA-12 detected by in vitro homocysteinylation assays. For in vitro homocysteinylation, purified proteins were incubated with Hcy followed by LC-MS/MS analysis to determine homocysteinylation sites. The homocysteinylated AA residues are shown in red, and the corresponding peptides identified by LC-MS/MS are listed. (E–H) Homocysteinylated AA residues in VHA-12 immunoprecipitated from cbs-1(yq357) mutants carrying a single-copy insertion of VHA-12::GFP (yqSi11). Worms were fed on OP50 E. coli. VHA-12::GFP was immunoprecipitated from yqSi11;cbs-1(yq357) animals and subjected to LC-MS/MS analysis to determine homocysteinylation sites. Left: LC-MS/MS spectra of peptides containing homocysteinylation sites. Right: relative abundance of homocysteinylation at the sites shown on the left. LC-MS/MS–detected peptides with homocysteinylation at K14 (E), K90 (F), C269 (G), and K437 (H) are shown. The x and y axes represent m/z and relative ion intensity, respectively. For all quantifications, statistical significance was calculated using the two-sided Student’s t test. **P < 0.01; ***P < 0.001. Source data are available for this figure: SourceData F5.