(A) Representative images of 293T cells transfected with GFP-fused VepA or VepAΔC. Actin was stained with rhodamine-phalloidin to visualize the cells. Scale bar, 200 µm. (B) The indicated proteins were delivered into HeLa cells using the HVJ envelope, or HeLa cells were treated with 100 µg ml⁻¹ VepA without HVJ; cytotoxicity was determined after 4 h. The values represent the mean ± SD for a minimum of three independent experiments. (C) HeLa cells were infected with the indicated strains for 4 h and analyzed for lactate dehydrogenase (LDH) release as a measure of cytotoxicity. *P<0.01. (D) Secretion of VepA or VepAΔC. The indicated strains were grown in LB broth for 6 h, and the secreted proteins were subjected to immunoblot analysis using an anti-VepA antibody. The migration positions of the molecular weight markers are indicated on the left side of the panel.

(A) S. cerevisiae BY4730 was transformed with the vector p426 or plasmids encoding wild-type VepA or VepAΔC. Then, 10-fold serially diluted cultures of yeast harboring each plasmid were spotted onto glucose (Glc) or galactose (Gal) plates and incubated for 72 h. (B) Scheme of the genome-wide screen for yeast knockout (YKO) strains resistant to VepA. YKO strains were grown individually in YPD broth in 96-well plates. For each plate, the strains were grown, pooled into a single culture and transformed with p426-VepA. The transformants were plated on SC plates lacking uracil and containing galactose (SC-Ura+Gal). After incubation at 30°C for 3 days, isolated colonies were analyzed to identify strains as described in Materials and Methods. (C) The growth patterns of the 10-fold serial dilutions of the Δvma3 strains harboring p426, p426-VepA, p426-VopT or p426-VopP on Glc and Gal plates are shown. (D) Immunoblot analysis using an anti-VepA antibody against lysates from Δvma3 strains harboring p426 or p426-VepA grown in Glc or Gal. (E) The sensitivity of V-ATPase mutants to VepA. A total of 14 yeast V-ATPase mutants (V₁ domain; A, B, C, D, E, F, G, H, V₀ domain; a, c, c′, c″, d, e) were transformed with p426-VepA. Then 10-fold dilution cultures of each mutant harboring p426-VepA were spotted onto SC-Ura+Glc or SC-Ura+Gal plates and incubated for 3 days at 30°C.

(A) HeLa cells treated with siRNAs were infected with the POR-3 (black bars) or ΔvepA strain (white bars) for 4 h, and cytotoxicity was measured using the LDH release assay. Data represent the mean ± SD. *P<0.01. (B) The knockdown level of the expression of the indicated proteins was confirmed by immunoblotting. (C) VepA translocation. HeLa cells treated with siRNAs were infected with the indicated strain for 3 h, and cell lysates were analyzed by immunoblotting with an anti-VepA antibody. An immunoblot for actin is shown as a loading control. (D) HeLa cells were left untreated or pre-treated with bafilomycin A (BafA) or concanamycin A (ConA) for 1 h and subsequently infected with POR-3 for 4 h. Cytotoxicity was determined using the LDH assay.

(A) Pull-down assays with biotinylated VepA (b-VepA) or b-VepAΔC from lysates of 293T cells expressing ATP6V0C-Flag. Bound proteins were subjected to immunoblot analysis using an anti-Flag antibody. (B) Endogenous proteins of RAW264.7 cells that were pulled down with b-VepA or b-VepAΔC were separated by SDS-PAGE and detected by silver staining. Arrows indicate the locations of VepA, VepAΔC, streptavidin and ATP6V0C.

(A–I) AO relocation assays. HeLa cells were exposed to AO and then infected with the indicated strains for 3 h. AO fluoresces red in lysosomes and green in the cytosol. (D, E, F) High magnification of the area marked by white boxes in A, B and C. Scale bar, 20 µm. (J) Quantification of the green fluorescence intensity from the experiments shown in Figure 5A–G. (K) Cytosolic extracts of HeLa cells infected with the indicated strains for 3 h were subjected to immunoblot analysis using an anti-CatD antibody. Immunoblots for Hsp90 were performed as loading controls. Lysis represents the total release of CatD. (L) HeLa cells that were treated with siControl or siV0C#2 were infected with the indicated strains for 3 h, and cytosolic extracts were subjected to immunoblot analysis using an anti-CatD antibody. (M) Cell-free lysosome preparations were treated with VepA or VepAΔC for 2 h, centrifuged to pellet the lysosomes (p) and separate the supernatant (s) and subjected to immunoblot analysis for CatD. Immunoblots for Lamp-1, a lysosomal integral membrane protein, were performed as loading controls. Treating lysosomes with 1% Triton X-100 (TX-100) results in total lysis and was used as a positive control. Whole cell lysates (WCL) and lysosome preparations (LY) are also shown. (N) The effect of ATP6V0C knockdown on VepA-induced lysosomal rupture in a cell-free system. Lysosomes prepared from HeLa cells treated with siControl or siV0C#2, were left untreated (UT) or were treated with TX-100 or VepA. After pelleting the lysosomes (p) and separating the supernatant (s), both fractions were subjected to immunoblot analysis for CatD or Lamp-1.