The c subunit of the Escherichia coli F-0 has been tagged with a hexahistid
ine motif at its C-terminus. The tagged subunit is capable of forming funct
ional F-0 complexes that translocate protons in the absence of the F-1 comp
lex. In the presence of F-1, the two sectors associate and display all bioc
hemical activities of the wildtype enzyme: DCCD-inhibitable ATPase activity
, ATP synthase activity, and ATP-dependent proton pumping. The enzyme can b
e solubilized and purified as an intact complex under native conditions on
immobilized-metal affinity chromatography (IMAC) resin. The purified comple
x can be reincorporated into liposomes and demonstrates ATP-dependent proto
n pumping activity. Hexahistine tags placed at the N-terminus, in contrast,
were all inactive. These experiments demonstrate the feasibility of taggin
g the c subunit for further studies of the F-0 and suggest an important rol
e for the N-terminus of the c subunit in either assembly or function of the
protein. (C) 2001 Academic Press.