title: Definition of membrane topology and identification of residues important for transport in subunit a of the vacuolar ATPase.
abstract: Subunit a of the vacuolar H(+)-ATPases plays an important role in proton transport. This membrane-integral 100-kDa subunit is thought to form or contribute to proton-conducting hemichannels that allow protons to gain access to and leave buried carboxyl groups on the proteolipid subunits (c, c', and c″) during proton translocation. We previously demonstrated that subunit a contains a large N-terminal cytoplasmic domain followed by a C-terminal domain containing eight transmembrane (TM) helices. TM7 contains a buried arginine residue (Arg-735) that is essential for proton transport and is located on a helical face that interacts with the proteolipid ring. To further define the topology of the C-terminal domain, the accessibility of 30 unique cysteine residues to the membrane-permeant reagent N-ethylmaleimide and the membrane-impermeant reagent polyethyleneglycol maleimide was determined. The results further define the borders of transmembrane segments in subunit a. To identify additional buried polar and charged residues important in proton transport, 25 sites were individually mutated to hydrophobic amino acids, and the effect on proton transport was determined. These and previous results identify a set of residues important for proton transport located on the cytoplasmic half of TM7 and TM8 and the lumenal half of TM3, TM4, and TM7. Based upon these data, we propose a tentative model in which the cytoplasmic hemichannel is located at the interface of TM7 and TM8 of subunit a and the proteolipid ring, whereas the lumenal hemichannel is located within subunit a at the interface of TM3, TM4, and TM7.
authors.affiliation: Graduate Program in Cell and Molecular Physiology, Sackler School of Graduate Biomedical Sciences, School of Medicine, Tufts University, Boston, Massachusetts 02111, USA.
authors.forename: Masashi
authors.initials: M
authors.lastname: Toei
authors.forename: Satoko
authors.initials: S
authors.lastname: Toei
authors.forename: Michael
authors.initials: M
authors.lastname: Forgac
mesh_terms: D001665:Binding Sites; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D003593:Cytoplasm / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology; D005656:Fungal Proteins / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010641:Phenotype; D017434:Protein Structure, Tertiary; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics*
chemical_list: D005656:Fungal Proteins; D025262:Vacuolar Proton-Translocating ATPases; D003545:Cysteine; D005033:Ethylmaleimide