Analysis of the membrane topology of transmembrane segments in the C-terminal hydrophobic domain of the yeast vacuolar ATPase subunit a (Vph1p) by chemical modification

Abstract

The integral V(0) domain of the vacuolar (H(+))-ATPases (V-ATPases) provides the pathway by which protons are transported across the membrane. Subunit a is a 100-kDa integral subunit of V(0) that plays an essential role in proton translocation. To better define the membrane topology of subunit a, unique cysteine residues were introduced into a Cys-less form of the yeast subunit a (Vph1p) and the accessibility of these cysteine residues to modification by the membrane permeant reagent N-ethylmaleimide (NEM) and the membrane impermeant reagent polyethyleneglycol maleimide (PEG-mal) in the presence and absence of the protein denaturant SDS was assessed. Thirty Vph1p mutants containing unique cysteine residues were constructed and analyzed. Cysteines introduced between residues 670 and 710 and between 807 and 840 were modified by PEG-mal in the absence of SDS, indicating a cytoplasmic orientation. Cysteines introduced between residues 602 and 620 and between residues 744 and 761 were modified by NEM but not PEG-mal in the absence of SDS, suggesting a lumenal orientation. Finally, cysteines introduced at residues 638, 645, 648, 723, 726, 734, and at nine positions between residue 766 and 804 were modified by NEM and PEG-mal only in the presence of SDS, consistent with their presence within the membrane or at a protein-protein interface. The results support an eight transmembrane helix (TM) model of subunit a in which the C terminus is located on the cytoplasmic side of the membrane and provide information on the location of hydrophilic loops separating TM6, 7, and 8.

FIGURE 1.

Analysis of stability of V-ATPase complexes containing single cysteine mutants of Vph1p by Western blot of isolated vacuolar membranes using antibodies against subunit a (part of V₀) and subunit A (part of V₁). Vacuolar membranes were isolated from the yeast strain MM112 (disrupted in the endogenous VPH1 and STV1 genes) expressing either a wild-type form of Vph1p (WT), the pRS316 vector alone (Vector), the Cys-less form of Vph1p (cysless) or the indicated single cysteine-containing mutants of Vph1p. Samples of vacuolar membranes (2 μg of protein) were separated by SDS-PAGE followed by transfer to nitrocellulose and Western blot using mouse monoclonal antibodies against subunit a (10D7) or subunit A (8B1-F3) as described under “Experimental Procedures.”

FIGURE 2.

Effect of mutations in Vph1p on concanamycin-sensitive ATPase activity and concanamycin-sensitive, ATP-dependent proton transport in isolated vacuolar membranes. Vacuolar membranes were isolated from the yeast strain MM112 (disrupted in the endogenous VPH1 and STV1 genes) expressing either a wild-type form of Vph1p (WT), the pRS316 vector alone (Vector), the Cys-less form of Vph1p (Cysless) or the indicated single cysteine-containing mutants of Vph1p. ATPase activity (solid bars) was measured using a continuous spectrophotometric assay, and ATP-dependent proton transport (open bars) was measured as the rate of change of fluorescence using the pH-sensitive dye ACMA in the presence or absence of 1μm concanamycin as described under “Experimental Procedures.” Values are expressed relative to vacuolar membranes expressing wild-type Vph1p. Concanamycin-sensitive ATPase activity for wild-type vacuolar membranes was 1.12 μg/min/mg protein. Values represent the average of at least two measurements on two independent vacuolar membrane preparations, with the error bars corresponding to the S.E.

FIGURE 3.

Theoretical Western blot pattern predicted for the cysteine accessibility assay. Vph1p mutants containing single cysteine residues either exposed to the cytoplasm (left panel), sequestered within the membrane (center panel), or exposed to the lumen (right panel) of sealed vacuolar membranes are predicted to show the indicated labeling patterns following sequential treatment in the presence or absence of NEM, the presence or absence of SDS and the presence of PEG-mal. Cysteines exposed to the cytoplasmic surface are modified by PEG-mal (and hence show a shift to lower mobility) in the absence of SDS and NEM but do not show this shift on pretreatment with NEM. Cysteines buried within the membrane (or otherwise shielded from modification) cannot be modified by either PEG-mal or NEM in the absence of SDS. Cysteines exposed on the lumenal surface of the membrane cannot be modified by PEG-mal in the absence of SDS but can be modified by NEM in the absence of SDS, resulting in pretreatment with NEM preventing the shift in mobility observed with PEG-mal in the presence of SDS.

FIGURE 4.

Modification of Vph1p mutants containing single cysteine residues by PEG-mal following sequential treatment in the presence or absence of NEM and the presence or absence of SDS. Vacuolar membranes were isolated from the yeast strain MM112 (disrupted in the endogenous VPH1 and STV1 genes) expressing either the Cys-less form of Vph1p (Cysless) or the indicated single cysteine-containing mutants of Vph1p. 200 μg of vacuolar membrane protein were split into two samples and treated sequentially in the presence or absence of NEM, the NEM removed, and the samples split again and treated in the presence or absence of SDS followed finally by treatment with PEG-mal, separation of samples on SDS-PAGE and Western blotting using the monoclonal antibody 10D7 against Vph1p as described under “Experimental Procedures.” Modification of Vph1p by PEG-mal results in a shift in a portion of the Vph1p band by 5–10 kDa. Panel a, the Cys-less Vph1p and cysteine mutants of Vph1p showing a cytoplasmic labeling pattern (see Fig. 3). Panel b, cysteine mutants of Vph1p showing a labeling pattern consistent with localization in a transmembrane segment or in some other location inaccessible to modification by either NEM or PEG-mal in the absence of SDS. Panel c, cysteine mutants of Vph1p showing a lumenal labeling pattern.

FIGURE 5.

Topological model of the yeast subunit a (Vph1p). Folding diagram of the yeast V-ATPase subunit a (Vph1p) based upon previous data (15), hydropathy analysis, and the results presented in the current work. Residues shown in open squares correspond to those cysteines showing a cytoplasmic labeling pattern (Fig. 4a). Residues shown in open circles correspond to cysteines whose labeling pattern is consistent with their presence in a transmembrane segment or in a location which prevents their reaction with NEM or PEG-mal in the absence of SDS (Fig. 4b). Residues shown in black boxes correspond to cysteines showing a lumenal labeling pattern (Fig. 4c). Residues shown in shaded boxes correspond to residues previously shown to have a cytoplasmic orientation (15). Residues shown in shaded circles correspond to buried charged residues whose mutation reduces (but does not eliminate) ATP-dependent proton transport by the V-ATPase. Arg-735 (shown as a black circle) corresponds to the buried arginine residue that is essential for ATP-dependent proton transport. Also shown is the site at position 560 where introduction of a factor Xa cleavage site resulted in sensitivity of the mutant Vph1p to cleavage by factor Xa protease from the cytoplasmic side of the membrane (15). The resulting model shows eight transmembrane segments with both the N and C terminus of the protein exposed to the cytoplasmic side of the membrane.