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.
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.
Department of Physiology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111, USA.
Yanru
Y
Wang
Masashi
M
Toei
Michael
M
Forgac
D002462:Cell Membrane / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D003593:Cytoplasm / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D015966:Gene Expression Regulation, Fungal; D057927:Hydrophobic and Hydrophilic Interactions; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010641:Phenotype; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry / Q000502:physiology*
D003902:Detergents; D025262:Vacuolar Proton-Translocating ATPases; D003545:Cysteine; D005033:Ethylmaleimide