Analysis of VHA-12 localization and VHA-12 mutations affecting the homocysteinylated residues. (A) Colocalization of VHA-12::GFP with VHA-5::RFP to MVBs/lysosomes but not 2xFYVE::mK2 (early endosomes) in the hypodermis in WT animals (2 days after L4). Bars, 5 μm. (B) Overexpression of VHA-12(K14A)::GFP and VHA-12(K437A)::GFP 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 overexpression of VHA-12(K14A)::GFP and VHA-12(K437A)::GFP are shown on the left. Bars, 5 μm. Quantification of lysosomes is shown on the right. ≥20 lysosomes from 3 animals (2 days after L4) were examined for each genotype. Data (mean ± SEM) are from three independent experiments. ****P < 0.0001.