Helical interactions and membrane disposition of the 16-kDa proteolipid subunit of the vacuolar H(+)-ATPase analyzed by cysteine replacement mutagenesis.
Theoretical mechanisms of proton translocation by the vacuolar H(+)-ATPase require that a transmembrane acidic residue of the multicopy 16-kDa proteolipid subunit be exposed at the exterior surface of the membrane sector of the enzyme, contacting the lipid phase. However, structural support for this theoretical mechanism is lacking. To address this, we have used cysteine mutagenesis to produce a molecular model of the 16-kDa proteolipid complex. Transmembrane helical contacts were determined using oxidative cysteine cross-linking, and accessibility of cysteines to the lipid phase was determined by their reactivity to the lipid-soluble probe N-(1-pyrenyl)maleimide. A single model for organization of the four helices of each monomeric proteolipid was the best fit to the experimental data, with helix 1 lining a central pore and helix 2 and helix 3 immediately external to it and forming the principal intermolecular contacts. Helix 4, containing the crucial acidic residue, is peripheral to the complex. The model is consistent not only with theoretical proton transport mechanisms, but has structural similarity to the dodecameric ring complex formed by the related 8-kDa proteolipid of the F(1)F(0)-ATPase. This suggests some commonality between the proton translocating mechanisms of the vacuolar and F(1)F(0)-ATPases.
Harrison
M A
MA
School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom. m.a.harrison@leeds.ac.uk
Murray
J
J
Powell
B
B
Kim
Y I
YI
Finbow
M E
ME
Findlay
J B
JB
D002462:Cell Membrane / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011510:Proteolipids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae
D011510:Proteolipids; D006180:Proton-Translocating ATPases; D003545:Cysteine