Hcy accumulation accounts for lysosomal dysfunction in C. elegans cbs-1 mutants. (A) Schematic representation of the Hcy metabolism pathway. Human enzymes and C. elegans genes encoding corresponding homologs are listed at each step. MAT1/2A, Met adenosyltransferase 1A/2A; GNMT, glycine N-methyltransferase; AHCY, adenosylhomocysteinase; CTH, cystathionine gamma-lyase; SAH, S-adenosyl Hcy; Cys, cysteine; THF, tetrahydrofolate. (B) Hcy content in whole animals and lysosomes of WT and cbs-1(yq357) mutants grown on E. coli OP50 (left). Lysosomes in WT and cbs-1(yq357) animals (2 days after L4) expressing LMP-1::RFP::3×HA were purified with HA-beads and examined with western blotting using mCh antibody (right). Tubulin was used as cytosolic protein control. ≥1,000 animals were used to measure the whole animal Hcy for each genotype, and ≥2,000 animals were used to isolate lysosomes for measurement of lysosomal Hcy for each genotype. Data (mean ± SEM) are from three independent experiments. (C) Hcy content in WT, cbs-1(yq357), sams-1(tm3945) single, and cbs1(yq357) sams-1(tm3945) double mutants grown on E. coli OP50. ≥1,000 animals were used to measure the Hcy concentration for each genotype. Data (mean ± SEM) are from three independent experiments. (D) Loss of sams-1 function suppresses lysosomal enlargement and shortened body length in cbs-1(yq357) mutants. Left: DIC images of adult worms (2 days after L4) and NUC-1::mCh-labeled lysosomes in the hypodermis of cbs-1(yq357) single mutant and cbs-1(yq357) combined with different sams-1 alleles (yq547, yq548, and tm3945). Bars, 200 μm(left) and 5 μm(right). Right: quantifications of lysosomal sizes (≥20 lysosomes from 3 animals for each genotype) and body length (≥15 animals for each genotype). (E) Hcy levels in cbs-1(yq357) mutants grown on OP50 E. coli without or with VB12 supplementation. ≥1,000 animals were used to measure the Hcy concentration for each group. Data (mean ± SEM) are from three independent experiments. (F) VB12 ameliorates lysosomal enlargement and shortened body length in cbs-1(yq357) mutants. Left: images of NUC-1::mCh-labeled lysosomes in the hypodermis (top) of cbs-1(yq357) animals and DIC images (bottom) of corresponding adults (2 days after L4) grown on E. coli OP50 without or with VB12 supplementation. Bars, 5 μm(top) and 200 μm(bottom). Middle: quantifications of lysosomal sizes (≥20 lysosomes from 3 animals for each genotype). Right: quantification of body length (≥15 animals for each genotype). Data (mean ± SEM) are from three independent experiments. For quantifications, data are presented as mean ± SEM. Statistical comparisons were performed using the two-sided Student’s t test. **P < 0.01, ***P < 0.001, and ****P < 0.0001. DIC, differential interference contrast. Source data are available for this figure: SourceData F3.