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Fig. 3. vph1{Delta} strain has an assembled V-ATPase complex in the vacuole. (A) Yeast vacuoles were isolated from wild type (WT), vph1{Delta} and stv1{Delta} strains as described in Materials and methods. Samples containing 10µg protein were applied in each lane. Following electrotransfer, the nitrocellulose membranes were decorated with the indicated antibodies. (B) Isolated yeast vacuoles of vph1{Delta} cells (0.5 ml) were placed on top of 20%-60% sucrose gradients in a buffer containing 20 mmol l-1 Mops, pH 7.2, 1 mmol l-1 EDTA. The gradients were centrifuged in an SW-40 rotor at 150,000 g for 14 h. The first 13 fractions collected from the bottom of the tube were assayed by immunoblotting for the V-ATPase with Vma8p and Stv1p, an endosomal t-SNARE Pep12p, and a vacuolar protein CPY. In both fractions 8 and 13, the degradation product of Stv1p, as reported earlier (Manolson et al., 1994) is visible.