Hcy binds to and homocysteinylates the V-ATPase subunit VHA-13. (A) Left: Coomassie blue staining of purified His₆-VHA-13 and His₆-VHA-9. Right: binding of Hcy to His₆-VHA-13 and His₆-VHA-9 in MST assays. Data represent the mean of three independent experiments. (B) Summary of homocysteinylation sites in VHA-13 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 AAs are shown in red, and the corresponding peptides identified by LC-MS/MS are listed. (C–G) Homocysteinylated AA residues in VHA-13 co-immunoprecipitated with VHA-12::GFP from cbs-1(yq357) mutants expressing single-copy insertion of VHA-12::GFP (yqSi11) as shown in Fig. 5. Worms were fed on OP50 E. coli. Endogenous VHA-13, coprecipitated with VHA-12::GFP, was subjected to LC-MS/MS analysis. 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 K96 (C), C243 (D), K245 (E), K382/C383 (F), and K574 (G) are shown. The x and y axes represent m/z and relative ion intensity, respectively. Source data are available for this figure: SourceData FS3.