title: Reconstitution of Fo of the sodium ion translocating ATP synthase of Propionigenium modestum from its heterologously expressed and purified subunits.
abstract: The atpB and atpF genes of Propionigenium modestum were cloned as His-tag fusion constructs and expressed in Escherichia coli. Both recombinant subunits a and b were purified via Ni(2+) chelate affinity chromatography. A functionally active Fo complex was reassembled in vitro from subunits a, b and c, and incorporated into liposomes. The F(o) liposomes catalysed (22)Na(+) uptake in response to an inside negative potassium diffusion potential, and the uptake was prevented by modification of the c subunits with N,N'-dicyclohexylcarbodiimide (DCCD). In the absence of a membrane potential the Fo complexes catalysed (22)Na(+)(out)/Na(+)(in)-exchange. After F(1) addition the F(1)F(o) complex was formed and the holoenzyme catalysed ATP synthesis, ATP dependent Na(+) pumping, and ATP hydrolysis, which was inhibited by DCCD. Functional F(o) hybrids were reconstituted with recombinant subunits a and b from P. modestum and c(11) from Ilyobacter tartaricus. These Fo hybrids had Na(+) translocation activities that were not distinguishable from that of P. modestum F(o).
authors.affiliation: Institut für Mikrobiologie, Eidgenössische Technische Hochschule, Zürich, Switzerland.
authors.forename: Franziska
authors.initials: F
authors.lastname: Wehrle
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Appoldt
authors.forename: Georg
authors.initials: G
authors.lastname: Kaim
authors.forename: Peter
authors.initials: P
authors.lastname: Dimroth
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli; D006089:Gram-Negative Anaerobic Bacteria / Q000201:enzymology*; D017136:Ion Transport; D006180:Proton-Translocating ATPases / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D012964:Sodium; D006180:Proton-Translocating ATPases