J Biol Chem, Vol. 274, Issue 48, 33999-34004, November 26, 1999

The Na⁺-F₁F₀-ATPase Operon from Acetobacterium woodii
OPERON STRUCTURE AND PRESENCE OF MULTIPLE COPIES OF atpE WHICH ENCODE PROTEOLIPIDS OF 8- AND 18-kDa

Stefan Rahlfs, Sascha Aufurth^§, and Volker Müller^§

From the  Institut für Mikrobiologie und Genetik der Georg-August-Universität, Grisebachstrasse 8, 37077 Göttingen, Germany and the ^§ Lehrstuhl für Mikrobiologie der Ludwig-Maximilians-Universität, Maria-Ward-Strasse 1a, 80638 München, Germany

Eight genes (atpI, atpB, atpE₁, atpE₂, atpE₃, atpF, atpH, and atpA) upstream of and contiguous with the previously described genes atpG, atpD, and atpC were cloned from chromosomal DNA of Acetobacterium woodii. Northern blot analysis revealed that the eleven atp genes are transcribed as a polycistronic message. The atp operon encodes the Na⁺-F₁F₀-ATPase of A. woodii, as evident from a comparison of the biochemically derived N termini of the subunits with the amino acid sequences deduced from the DNA sequences. The molecular analysis revealed that all of the F₁F₀-encoding genes from Escherichia coli have homologs in the Na⁺-F₁F₀-ATPase operon from A. woodii, despite the fact that only six subunits were found in previous preparations of the enzyme from A. woodii. These results unequivocally prove that the Na⁺-ATPase from A. woodii is an enzyme of the F₁F₀ class. Most interestingly, the gene encoding the proteolipid underwent quadruplication. Two gene copies (atpE₂ and atpE₃) encode identical 8-kDa proteolipids. Two additional gene copies were fused to form the atpE₁ gene. Heterologous expression experiments as well as immunolabeling studies with native membranes revealed that atpE₁ encodes a duplicated 18-kDa proteolipid. This is the first demonstration of multiplication and fusion of proteolipid-encoding genes in F₁F₀-ATPase operons. Furthermore, AtpE₁ is the first duplicated proteolipid ever found to be encoded by an F₁F₀-ATPase operon.