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Reference: Wang Y, et al. (2008) 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. J Biol Chem 283(30):20696-702

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Abstract


The integral V0 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 V0 that plays an essential role in proton translocation. In order 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 residue 602 and 620 and between residue 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.

Reference Type
Journal Article
Authors
Wang Y, Toei M, Forgac M
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