VMA6 rescued vph1-tether deletions mutants. A, exogenous subunit d rescued growth of vph1-Δ362–407 and vph1-Δ362–377 at neutral pH. Vph1Δstv1Δ cells were co-transformed with the VMA6 gene plus either wild-type (WT) VPH1 gene or VPH1 carrying the indicated truncation. Each gene was expressed from the low copy plasmid pRS316 under control of their natural promoter. Transformants were selected in SD−Ura plates adjusted to pH 5. Cell growth was compared at pH 5 and 7.5 on SD−Ura plates for 96 h at 30 °C. Controls included vph1Δstv1Δ transformed with wild-type VPH1 alone, VPH1-Δ362–407 alone, and VPH1-Δ362–377 alone. B, plasmid pRS316 alone cannot rescue pH-sensitive growth. Vph1Δstv1Δ cells were co-transformed with the plasmid pRS316 plus VPH1 carrying the indicated truncations at the tether. Controls included the wild-type VPH1 gene alone and the VMA6 gene alone, each in the plasmid pRS316. Transformants were selected in SD−Ura plates pH 5. Cell growth was compared on SD−Ura plates at pH 5 and 7.5 incubated for 96 h at 30 °C.

Tether-less mutant V₁V₀ is delivered to the vacuoles of cells transformed with exogenous subunit d. Left, vacuolar vesicles from vph1Δstv1Δ cells co-transformed with VMA6 plus either wild-type VPH1 or mutant VPH1 were purified by Ficoll gradient density centrifugation. Vacuolar protein (25 μg) solubilized in cracking buffer was loaded onto 10% polyacrylamide gels and separated by SDS-PAGE. Protein was transferred to nitrocellulose, and blots were probed with monoclonal antibodies against subunits a (HA), A, and B and polyclonal antibodies against subunits D, E, and d. Immunoblots were as described in the legend to Fig. 2. Right, Vacuolar membranes from either wild-type VPH1 or mutant VPH1 were purified by Ficoll gradient centrifugation. Vacuolar protein was solubilized and analyzed by immunoblotting using antibodies against V₀ subunits a and d and V₁ subunits A, B, D, and E. For loading control, membranes were probed with a monoclonal antibody against vacuolar alkaline phosphatase (ALK).

Exogenous subunit d allowed V₁V₀ assembly of tether deletion mutants. A, whole cell lysates. Whole cell lysates were prepared from vph1Δstv1Δ cells co-transformed with VMA6 plus either wild-type (WT) VPH1 or VPH1 carrying tether deletions. Each gene was expressed from the low copy plasmid pRS316 under control of their corresponding natural promoter. Cells were grown overnight to mid-log phase in SD−Ura pH 5. Protein extracts were prepared as described in the legend to Fig. 3A, except that a 10% gel was used. Protein was transferred to a nitrocellulose membrane for detection of subunits a, A, B, and d by Western blots as described in the legend to Fig. 2. B, quantitative immunoblotting of V-ATPase subunits in whole cell lysates. Serial dilutions of whole cell lysates prepared from wild-type and tether-less vph1 mutant cells co-transformed with VMA6 were prepared in cracking buffer. Whole cell lysates were prepared as described above. The indicated amounts of A₆₀₀ in each well were separated in 10% SDS-PAGE and analyzed by Western blotting. C, immunoprecipitation of V₁V₀. Vph1Δstv1Δ co-transformed with VMA6 plus either wild-type VPH1 or VPH1 carrying the indicated truncations was grown in SD−Ura medium at pH 5. Cells were converted to spheroplasts, lysed, and immunoprecipitated with anti-HA antibody as described in the legend to Fig. 2B. Immunoprecipitated protein was separated by SDS-PAGE (10% gel) and transferred to a nitrocellulose membrane for blotting using primary antibodies to V-ATPase subunits a (HA), A, and B. The band corresponding to the antibody heavy chain (HC) is indicated.

The tether at the N-terminal domain of Vph1 is necessary for V-ATPase function. A, N-terminal domain secondary structure prediction. Secondary structure prediction of the amino acid sequence at the N-terminal hydrophilic domain of subunit a Vph1 was performed using the PSIPRED Protein Structure Prediction Server of the University College London (35). Only the first transmembrane-spanning helix of the C-terminal domain is shown. Tether deletions made in this study are indicated (▵). Shown are the predicted α-helix (gray cylinders), β-sheet (black arrow), and coiled (black connecting line) structures. Location of the two copies of the ΗΑ sequence introduced after residue Asn-185 (28) are indicated by the box. B, growth phenotype of Vph1 tether deletion mutants. Vph1Δstv1Δ cells were transformed with the wild-type (WT) VPH1 gene or VPH1 carrying the indicated truncations at the tether. Wild-type and mutant VPH1 were expressed from the low copy plasmid pRS316. Serial dilutions (left to right: 10-, 10²-, 10³- and 10⁴-fold) were performed into sterile water, and aliquots of each dilution were transferred to SD−Ura plates either at pH 5 or pH 7.5. Cells were grown for 96 h at 30 °C.

V₀(−d) domains are present in subunit a-tether deletion mutants. A, whole cell lysates. Vph1Δstv1Δ cells transformed with the wild-type (WT) VPH1 gene or VPH1 carrying the indicated truncations were grown overnight to mid-log phase in SD−Ura pH 5. A culture volume corresponding to Abs_{600 nm} = 20–30 from each strain was removed and lysed as described under “Experimental Procedures.” Proteins in the whole cell lysate (1–2 A₆₀₀ of the original culture) were loaded onto a 12% polyacrylamide gel and separated by SDS-PAGE. Extracts were blotted with a polyclonal antibody against subunit d, and monoclonal antibodies against subunits a (HA), A, and B. B, immunoprecipitation of biosynthetically labeled V-ATPase. Vph1Δstv1Δ transformed with the wild-typeVPH1 gene and VPH1 carrying tether truncations were grown to mid-log phase in minimal medium lacking methionine and the nutritional marker uracil (SD−Met-Ura pH 5). Cells were converted to spheroplasts and biosynthetically labeled with Tran³⁵S-label as described under “Experimental Procedures.” Labeled spheroplasts were lysed, and V-ATPase immunoprecipitated under nondenaturing conditions using an antibody against HA. Immunoprecipitates were separated by SDS-PAGE using 13% gels, scanned in a Fuji Scanner 5100, and analyzed using the Multi Gauge software. V₁V₀ and V₀ complexes radiolabeled with ³⁵S were included in the same gel and used as reference for identifying V-ATPase subunits (left two lanes).

Glucose-controlled disassembly and reassembly of V₁V₀. A, pulse-chased experiments followed by V₁V₀ immunoprecipitation. Wild-type yeast cells were converted to spheroplasts and labeled with Tran³⁵S-label in SD−Met−Ura pH 5. Excess unlabeled cysteine and methionine were added to the radiolabeled spheroplasts, which were immediately resuspended in chase medium containing 1.2 m sorbitol and chased at 30 °C as follows: 20 min in YEPD (2% glucose), 10 min in YEP (no glucose), 10 min in YEP followed by an additional 10 min with 2% glucose added. A monoclonal antibody against subunit A of V₁ (8B1) was used for the immunoprecipitations. Immunoprecipitates were analyzed as described in the legend to Fig. 3B. V₁ subunits A and B and V₀ subunit a are indicated at the left. B, the glycolytic enzyme phosphofructokinase associates with V-ATPase. Left, vacuolar membranes were purified from wild-type (WT) and tether-less mutants (vph1-Δ362–407 and vph1-Δ362–377), each co-transformed with exogenous subunit d as described in the legend to Fig. 6. Shown is a Western blot of vacuolar membrane protein (25 μg) proved with a polyclonal antibody against yeast phosphofructokinase (PFK). Right, subunit a was immunoprecipitated from pH-sensitive tether-less mutants (vph1-Δ362–407 and vph1-Δ362–377) and from mutant cells that have been rescued by exogenous subunit d (vph1-Δ362–407+Vma6 and vph1-Δ362–377+Vma6). Anti-HA was used for immunoprecipitations as described in the legend to Fig. 2. Immunoprecipitates (IP) were analyzed by Western blots by using a polyclonal antibody against yeast phosphofructokinase. The band corresponding to the antibody heavy chain (HC) is indicated.

Vph1 tether deletion mutants failed to assemble V₁V₀ complexes. A, immunoprecipitation (IP) of V₁ subunit A. Cultures of vph1Δstv1Δ cells transformed with the wild-type (WT) VPH1 gene and VPH1 carrying the indicated truncations at the tether were converted to spheroplasts by treatment with zymolase. Spheroplasts were lysed in PBS containing protease inhibitors, 1% C₁₂E₉, and dithiobis[succinimidyl propionate]. Lysates were incubated with antibody 8B1 against V₁ subunit A, and V-ATPase subunits were immunoprecipitated after the addition of protein A-Sepharose as described under “Experimental Procedures.” A control which did not have the cell lysate added was incubated in parallel under the same conditions (antibody alone (Ab)). Immunoprecipitates were solubilized by the addition cracking buffer, subjected to SDS-PAGE using 12% gels, and analyzed by Western blots. Immunoblots were proved with monoclonal antibodies to V₁ subunits A and B and V₀ subunit a (anti-HA). Alkaline phosphatase-conjugated goat anti-mouse secondary antibody was added to blots which were developed by the addition of the alkaline-phosphatase substrates nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate. Bands corresponding to V-ATPase subunits a (100 kDa), A (69 kDa), and B (60 kDa) and the antibody heavy chain (HC) are indicated. B, immunoprecipitation of V₀ subunit a. Vph1Δstv1Δ cells transformed with the wild-type VPH1 gene and VPH1 with truncations at the tether were converted to spheroplasts and lysed as described under “Experimental Procedures.” V₀ subunit a was immunoprecipitated by using antibody 10D7 (left) and anti-HA (right). Immunoprecipitations were as described in the legend to A. Immunoprecipitates were analyzed by Western blots using antibodies to V₁ subunits A and B and V₀ subunits a (anti-HA) and d. V-ATPase subunits a (100 kDa), A (69 kDa), B (60 kDa), and d (36 kDa), and the antibody heavy and light chains (HC and LC) are indicated.