Mitochondrial ATP synthase (F(1)Fo-ATPase) catalyzes the terminal step of oxidative phosphorylation. In this paper, we demonstrate the functional expression of the hexahistidine-tagged beta-subunit of yeast ATP synthase and the purification of the F-1-ATPase from yeast cells. A gene encoding the beta-subunit from Saccharomyces cerevisiae was modified to encode a protein of which the original N-terminus import signal sequence was replaced by a sequence containing the import signal sequence of a mitochondrial ATPase inhibitor, its processing site, and six consecutive histidines. Expression of the modified gene generated a functional F(1)Fo complex in host yeast cells lacking a functional copy of the endogenous ATP2 gene, as judged by growth of rescued cells on lactate medium. F-1 was extracted from the yeast mitochondria by chloroform treatment and purified by immobilized metal affinity chromatography and gel filtration chromatography. The specific activity of the purified F-1 was comparable to that of the wild-type enzyme, and the F-1 contained all of the 5 known subunits (alpha, beta, gamma, delta, and epsilon).(1) Moreover, the activity of the F-1 was completely inhibited by the specific ATPase inhibitor protein, IF1. These results indicate that F-1 containing the tagged beta-subunit is fully assembled and active. The application of this novel procedure simplifies the number of steps required for the isolation of F-1 used for studying the molecular mechanism of catalysis and regulation of the enzyme. (C) 2004 Elsevier Inc. All rights reserved.