Reconstitution of Fo of the sodium ion translocating ATP synthase of Propionigenium modestum from its heterologously expressed and purified subunits.
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).
Wehrle
Franziska
F
Institut für Mikrobiologie, Eidgenössische Technische Hochschule, Zürich, Switzerland.
Appoldt
Yvonne
Y
Kaim
Georg
G
Dimroth
Peter
P
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*
D011993:Recombinant Fusion Proteins; D012964:Sodium; D006180:Proton-Translocating ATPases