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doi: 10.1242/10.1242/jcs.00328

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Role of the Vtc proteins in V-ATPase stability and membrane trafficking

Friedrich-Miescher-Laboratorium der Max-Planck-Gesellschaft, Spemannstr. 37-39, 72076 Tübingen, Germany

View larger version (76K): [in a new window]   Fig. 1. Membrane association of Vtc proteins. (A) Vtc1p and Vtc4p behave as integral membrane proteins. 60 µg of vacuoles from strain OMY1 in 1 ml of PS were centrifuged (10,000 g for 5 minutes at 4°C) and resuspended in 0.2 ml PS with one of the following additions: 100 mM KCl, 50 mM KOAc (low salt); 1.6 M KCl (high salt); 4 M urea; or 0.1 M Na2CO3. After 10 minutes at 30°C (or 30 minutes on ice for carbonate extraction), the samples were centrifuged (125,000 g, 20 minutes, 4°C). Pellets (P) were resuspended in 1 ml of PS and the supernatants (S) supplemented with PS ad 1 ml. All samples were TCA precipitated and analyzed by SDS-PAGE and western blotting. (B) Strains BJ3505 (pep4–) and DKY6281 (PEP4+) expressing a Vtc1p-GFP fusion were grown logarithmically in YPD and viewed under a confocal fluorescence microscope. Left panel: GFP fluorescence; right panel: Nomarski optics.

View larger version (32K): [in a new window]   Fig. 6. Levels of V-ATPase subunits in different deletion mutants. (A) 30 µg each of vacuoles from the indicated strains were precipitated with TCA and analyzed by SDS-PAGE and western blotting with the indicated antibodies. All strains were deficient for vacuolar proteases (pep4). Strain backgrounds were OMY1 (lanes 1-4) and SBY86 (lanes 5-6). (B) Same as in A, but with whole cell extracts.

View larger version (27K): [in a new window]   Fig. 2. Topology of Vtc proteins. (A) Protease digestion of vacuoles carrying Vtc1p-GFP or Vtc3p-GFP*. Vacuoles were isolated from BJ3505 cells expressing Vtc1p-GFP (from plasmid pYER-GFP) or from SBY593 cells expressing chromosomally encoded Vtc3p-GFP*. 20 µg vacuoles (0.1 mg/ml in PS buffer) were incubated with 10 µg/ml proteinase K in the presence or absence of 0.5% (w/v) Triton X-100 (5 minutes, 0°C). Digestion was stopped by adding one volume of 2 mM PMSF in PS buffer. Proteins were TCA precipitated, washed with acetone and solubilized in 100 µl non-reducing SDS-sample buffer. Coprecipitated Triton X-100 that can interfere with SDS-PAGE was extracted with chloroform/methanol (water:chloroform:methanol 2:1:2). Pellets were resolubilized in 50 µl reducing SDS-sample buffer, split and analyzed by 15% and 7.5% gels and western blotting with rabbit anti-GFP, goat anti-Vtc4p or mouse anti-Pho8p. TM, transmembrane fragment; pPho8p, pro-Pho8p; mPho8p, mature Pho8p; pPho8p-cytD, pPho8p fragment lacking the cytosolic tail. (B) Topology of the Vtc proteins and of Pho8p.

View larger version (66K): [in a new window]   Fig. 3. Biosynthetic and endocytic trafficking to the vacuole. (A) CPY transport was assayed by pulse chase as described previously (Peters et al., 1999), except that the pulse and chase were performed at 30°C. The strains used were OMY20 (vtc1::HIS3), OMY21 (vtc2::HIS3), OMY22 (vtc3::HIS3) and BY4727 (wt). The growth medium was supplemented with methionine (20 µg/ml). (B) Pulse-labeling with FM4-64: Cells were grown in YPD medium (12 hours, 25°C), labeled with 200 µM FM4-64 (2 minutes, 25°C), reisolated (30 seconds, 3000 g, 20°C), washed with YPD and reisolated as before. The cells were resuspended in YPD at OD600=1 and chased at 25°C for various times. The cells were reisolated (30 seconds, 3000 g, 20°C) and resuspended in the supernatant at OD600=10. 5 µl of the suspension were transferred to a microscopy slide and were quickly analyzed in a fluorescence microscope under minimal excitation.

View larger version (28K): [in a new window]   Fig. 4. Proton uptake and fusion of vacuoles. (A) Proton uptake of wild-type vacuoles (mixture of the fusion tester strains OMY1 and DKY6281) and vacuoles from vtc1 (OMY2/OMY5) was measured. (B) Proton uptake and fusion activity of wild-type vacuoles (OMY1/DKY6281 or SBY86/SBY85, respectively) were compared with those of vacuoles from vtc1 (OMY2/OMY5), vtc2 (OMY4/OMY7), vtc3 (OMY3/OMY6) and vtc4 (SBY83/SBY82). Proton uptake activity of the wild-type vacuoles was set to 100%. 100% wild-type control fusion was 3.85 U (OMY1/DKY6281) and 3.08 U (SBY86/SBY85), respectively. (C) Comparison of proton uptake activity and fusion activity of wild-type vacuoles (OMY1/DKY6281) in the presence of different concentrations of concanamycin A. Values were plotted as a percentage of the control (vacuoles without concanamycin A). n=3. Control fusions were 2.93 U, 2.40 U and 4.11 U. (D) Proton uptake and fusion activity of wild-type vacuoles (OMY1/DKY6281) with either control antibody or antibodies to Vtc4p. The antibody concentration was 60 µM (c.f. Muller et al., 2002). Proton uptake activity of the sample with control antibodies was set to 100%.

View larger version (48K): [in a new window]   Fig. 5. Proteolytic sensitivity of Vph1p depends on Vtc proteins. (A) 25 µg of vacuoles from the indicated strains expressing Vph1p with chromosomally encoded C-terminal tags were precipitated with TCA and analyzed by SDS-PAGE and western blotting with the indicated antibodies. All strains were derived from OMY1, that is, deficient for vacuolar proteases (pep4, prb1). (B) Same analysis as in A, but with whole cell extracts from 107 cells.

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