Functional expression of hexahistidine-tagged beta-subunit of yeast F1-ATPase and isolation of the enzyme by immobilized metal affinity chromatography.
Mitochondrial ATP synthase (F1Fo-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 F1-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 F1Fo complex in host yeast cells lacking a functional copy of the endogenous ATP2 gene, as judged by growth of rescued cells on lactate medium. F1 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 F1 was comparable to that of the wild-type enzyme, and the F1 contained all of the 5 known subunits (alpha, beta, gamma, delta, and epsilon). Moreover, the activity of the F1 was completely inhibited by the specific ATPase inhibitor protein, IF1. These results indicate that F1 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 F1 used for studying the molecular mechanism of catalysis and regulation of the enzyme.
Department of Food and Nutrition, Faculty of Human Life Science, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi, Osaka 558-8585, Japan. ichikawa@life.osaka-cu.ac.jp
Naoki
N
Ichikawa
Miki
M
Mizuno
D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D004791:Enzyme Inhibitors / Q000302:isolation & purification / Q000378:metabolism; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D008670:Metals / Q000737:chemistry*; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals / Q000235:genetics; D021122:Protein Subunits / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D000097795:ATPase Inhibitory Protein
D004791:Enzyme Inhibitors; D006639:Histidine; D008670:Metals; D021382:Protein Sorting Signals; D021122:Protein Subunits; D011506:Proteins; D006180:Proton-Translocating ATPases; D029701:Saccharomyces cerevisiae Proteins; D000097795:ATPase Inhibitory Protein; C471230:ATP2 protein, S cerevisiae