Fig. 3.

Vacuolar integrity tests. Intact vacuoles were isolated as described in Materials and Methods. Western blot was performed with anti-CPY antibodies (Upper) or anti-AtNHX1 (Lower). Vacuoles from yeast harboring the pHpl-2 plasmid were incubated with (lanes 2–5) or without (lane 1) 500 μg/ml proteinase K in the presence (lane 5) or absence (lanes 1–4) of Triton X-100. Untreated control vacuoles (lane 1) and vacuoles containing proteinase K and Triton X-100 (lane 5) were incubated for 90 min on ice. Vacuoles containing proteinase K only (lanes 2–4) were incubated for 30 min (lane 2), 60 min (lane 3), and 90 min (lane 4) on ice, respectively.