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Characterization of yeast V-ATPase mutants lacking Vph1p or Stv1p and the effect on endocytosis

Natalie Perzov, Vered Padler-Karavani, Hannah Nelson^* and Nathan Nelson

Department of Biochemistry, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel

View larger version (25K): [in a new window]   Fig. 1. Null mutation of VPH1 causes overexpression of Stv1p. (A) Western analysis of whole cell extracts of wild type (WT) and stv1 mutants. Stv1p is absent from the stv1 strain. Some non-specific bands are labeled by the polyclonal antibody. The positions of marker proteins (kDa) are shown. (B) Western analysis of wild type, vph1, stv1 and vma8 strains. Whole cell extracts from the strains were prepared as described in Materials and methods. Total cellular proteins (20 µg per lane) were subjected to SDS-PAGE and immunoblotted with antibodies against Stv1p, Vph1p and Vma8p.

View larger version (68K): [in a new window]   Fig. 2. Vph1p and Stv1p cofractionate with membranes containing the Golgi-endosome and vacuolar markers. An enriched Golgi membrane fractionation was prepared from the wild type as described in Materials and methods. Western analysis of these fractions with antibodies against Vph1p, Stv1p, Vma5p, Sec14p (a late Golgi protein), Sed5p (a cis-Golgi protein), Pep12p (an endosomal marker) and ALP (vacuolar membrane marker) is shown. Fraction numbers are indicated at the bottom.

View larger version (30K): [in a new window]   Fig. 3. vph1 strain has an assembled V-ATPase complex in the vacuole. (A) Yeast vacuoles were isolated from wild type (WT), vph1 and stv1 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 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.

View larger version (10K): [in a new window]   Fig. 4. Acridine Orange uptake into isolated vacuoles from stv1 and vph1 strains shows diminished activity. Yeast vacuoles were isolated by Ficoll gradient centrifugation and adjusted to a protein concentration of 2 mg ml-1 (Uchida et al., 1985). ATP-dependent proton uptake was measured by following Acridine Orange absorption changes (A) at 491-540 nm. Vacuoles containing 10 µg of protein were assayed in each sample. Where indicated by an arrow, 10 µl of 0.1 mol l-1 MgATP or 1 µl of 1 mmol l-1 carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) was added.

View larger version (109K): [in a new window]   Fig. 5. LysoSensor Green DND-189 is a vital dye that fluoresces in the acidic compartments of the yeast cell. (A) Comparison of LysoSensor Green DND-189 and Quinacrine staining. Wild-type cells were stained separately with LysoSensor Green DND-189 and Quinacrine as described in Materials and methods. (B) Wild-type cells were incubated at 0 °C for 30 min with 30 µmol l-1 FM 4-64 in YPD medium buffered with 100 mmol l-1 Hepes, pH 7.6. The cells were harvested by centrifugation, washed once with the same buffer, and resuspended in 0.35 ml of fresh medium containing 4 µmol l-1 LysoSensor Green DND-189. Cells were then incubated for 5 min, harvested by centrifugation, washed and resuspended in YPD medium. Portions were removed for microscopy and photographed at 25 min (top) and 45 min (bottom). Top left of each panel, FM 4-64 fluorescence; top right, the same cells in visible light; bottom left, LysoSensor Green DND-189 fluorescence; bottom right, the double-labeled image. The images were recorded, merged and processed using the Zeiss LSM Image Browser.

View larger version (69K): [in a new window]   Fig. 6. stv1 and vph1 strains show defects in vacuolar acidification. Wild-type (WT) and V-ATPase mutants were grown in YPD medium, pH 5.5, and stained with LysoSensor Green DND-189 as described in Materials and methods. Staining of vph1 and stv1/vph1 strains is shown at two different light intensities (*3 is triple intensity). The panel below shows staining of vph1 grown in YPD buffered at pH 7.5.

View larger version (60K): [in a new window]   Fig. 7. Differential effect of stv1 and vph1 mutants on Pma1p sorting and CPY and ALP processing. (A) Sucrose density gradients of whole cell extracts from wild type (WT), stv1, vph1 and stv1/vph1 strains were prepared as described in Materials and methods. The location of Pma1p was determined by immunoblot analysis with polyclonal antibody against purified Pma1p. Fraction numbers are indicated at the bottom. (B) Western analysis of vacuoles isolated from wild type, stv1 and vph1 strains by Ficoll gradient centrifugation and adjusted to a protein concentration of 2 mg ml-1 with antibodies against CPY and ALP. p, precursor of ALP; p1 and p2, precursors of CPY; m, mature form of the protein.

View larger version (72K): [in a new window]   Fig. 8. Endocytosis of the fluorescent endocytic marker FM 4-64 to vacuolar membranes is slowed down in V-ATPase null mutants. (A) Wild-type (WT) and vma3 cells were stained with FM 4-64 as described in Materials and methods. Samples were removed for microscopy after staining for the indicated times (min), in order to monitor the internalization kinetics of the dye. (B) The indicated strains were stained with FM 4-64 and photographed after a 60 min chase in YPD medium. Top panels show FM 4-64 fluorescence; bottom panels show visible light images of the same cells.

View larger version (58K): [in a new window]   Fig. 9. Concanamycin A inhibits endocytosis in wild-type cells. Wild-type cells were treated (+) or not (-) for 20 min with 3 µmoll-1 concanamycin A (dissolved in DMSO solution) before staining with FM 4-64, according to the standard procedure. Staining images were visualized at 50 min and 90 min. Left panels, FM 4-64 fluorescence; right panels, visible light images of the same cells.