Figure 1. CuCl₂ inhibits vacuole homotypic fusion.

Vacuoles isolated from wild type (BJ3505 and DKY6281) (A) or yvc1Δ (RFY74–75) (B) fusion reporter strains were incubated with buffer alone or a concentration curve of CuCl₂. (C) Wild type vacuoles were incubated with 100 μM MgCl₂, ZnCl₂, CoCl₂, CuCl₂, or CuSO₄. Fusion reactions were incubated for 90 min at 27°C. After incubation membranes were solubilized and incubated with p-nitrophenyl phosphate to measure Pho8 activity. p-nitrophenolate was measured at OD₄₀₀. Fusion values were normalized to the untreated control set to 1. (D) BJ3505 cells were grown to log phase and treated with CuCl₂. Vacuoles were visualized by staining with FM4–64 and cells were visualized using DIC. (E) Quantitation of vacuole fragmentation of cells treated with 0 μM or 50 μM CuCl₂ as in panel D. n=392, untreated cells, n=616, 50 μM CuCl2 treated cells. (F) DKY6281 vacuoles were incubated with 100 μM CuCl₂, 0.1% TX-100 or buffer for 90 min at 27°C. After incubation, the soluble and membrane fractions were separated by centrifugation (16,000 x g, 10 min, 4°C), mixed with SDS-loading buffer and resolved by SDS-PAGE. The soluble luminal protease Pep4 and the membrane anchored Ypt7 were probed for by immunoblotting. (G) Wild type vacuoles were treated with or without CuCl₂ in the presence of a concentration curve of PBN to eliminate oxygen radicals. Error bars are S.E.M. (n=3). Scale bar = 4 μm.