Na+ Transport by the A(1)A(O)-ATP Synthase Purified from Thermococcus onnurineus and Reconstituted into Liposomes

Abstract

The ATP synthase of many archaea has the conserved sodium ion binding motif in its rotor subunit, implying that these A(1)A(O)-ATP synthases use Na+ as coupling ion. However, this has never been experimentally verified with a purified system. To experimentally address the nature of the coupling ion, we have purified the A(1)A(O)-ATP synthase from T. onnurineus. It contains nine subunits that are functionally coupled. The enzyme hydrolyzed ATP, CTP, GTP, UTP, and ITP with nearly identical activities of around 40 units/mg of protein and was active over a wide pH range with maximal activity at pH 7. Noteworthy was the temperature profile. ATP hydrolysis was maximal at 80 degrees C and still retained an activity of 2.5 units/mg of protein at 45 degrees C. The high activity of the enzyme at 45 degrees C opened, for the first time, a way to directly measure ion transport in an A(1)A(O)-ATP synthase. Therefore, the enzyme was reconstituted into liposomes generated from Escherichia coli lipids. These proteoliposomes were still active at 45 degrees C. and coupled ATP hydrolysis to primary and electrogenic Na+ transport. This is the first proof of Na+ transport by an A(1)A(O)-ATP synthase and these findings are discussed in light of the distribution of the sodium ion binding motif in archaea and the role of Na+ in the bioenergetics of archaea.