Open AccessReconstitution of F o of the sodium ion translocating ATP synthase of Propionigenium modestum from its heterologously expressed and purified subunits
Author(s) -
Wehrle Franziska,
Appoldt Yvonne,
Kaim Georg,
Dimroth Peter
Publication year - 2002
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1033.2002.02923.x
Subject(s) - atp synthase , atp hydrolysis , protein subunit , liposome , recombinant dna , enzyme , sodium , chemistry , escherichia coli , biochemistry , potassium , stereochemistry , biology , atpase , gene , organic chemistry
The atp B and atp F 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 F o 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 F o 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 F o hybrids had Na + translocation activities that were not distinguishable from that of P. modestum F o .