FIGURE 3.
Pah1p is required for vacuole fusion. A, fusion reactions were carried out using WT and pah1Δ vacuoles or vacuoles from pah1Δ strains complemented with WT pPAH1 (B) or phosphatase dead pPAH1D398E (C). Fusion reactions were incubated at 27 °C for the indicated times and assayed for proPho8 maturation. Error bars represent S.E. (n = 3). D—G, yeast cells (WT, pah1Δ, pah1Δ + pPAH1, pah1Δ + pPAH1D398E) were incubated with 5 μm FM4-64 to label vacuoles. H, fusion was examined by a real-time lipid-mixing assay. WT or pah1Δ. Donor vacuoles were labeled with Rh-PE at self-quenching concentrations. Labeled donor vacuoles were incubated with the respective unlabeled acceptor vacuoles as described under “Experimental Procedures.” Fusion was measured by Rh-PE dequenching upon outer leaflet mixing. The experiment is representative of three trials. I–J, content mixing fusion assays were performed using vacuoles from WT, dpp1Δ (I) or lpp1Δ (J) strains. Errors bars represent S.E. (n = 3). Alk. Phos., alkaline phosphatase.