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 Ni2+ chelate affinity chromatography. A functionally active Fo complex was reassembled in vitro from subunits a, b and c, and incorporated into liposomes. The Fo liposomes catalysed 22Na+ 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 22Na+out/Na+in- exchange. After F1 addition the F1Fo complex was formed and the holoenzyme catalysed ATP synthesis, ATP dependent Na+ pumping, and ATP hydrolysis, which was inhibited by DCCD. Functional Fo hybrids were reconstituted with recombinant subunits a and b from P. modestum and c11 from Ilyobacter tartaricus. These Fo hybrids had Na+ translocation activities that were not distinguishable from that of P. modestum Fo.