A recombinant form of subunit E (Vma4p) from yeast vacuolar ATPases (V-ATPases) has been overexpressed in Escherichia coli, purified to homogeneity, and explored by mass spectrometry. Analysis of the secondary structure of Vma4p by circular dichroism spectroscopy indicated 32% α-helix and 23% β-sheet content. Vma4p formed a hybrid-complex with the nucleotide-binding subunits α and β of the closely related F₁ ATPase of the thermophilic bacterium PS3 (TF₁). The α₃β₃E-hybrid-complex had 56% of the ATPase activity of the native TF₁. By comparison, anα₃β₃-formation without Vma4p showed about 24% of total TF₁ ATPase activity. This is the first demonstration of a hydrolytically active hybrid-complex consisting of F₁ and V₁ subunits. The arrangement of subunit E in V₁ has been probed using the recombinant Vma4p, the α₃β₃E-hybrid-complex together with V₁ and an A₃B₃HEG-subcomplex of the V₁ ATPase from Manduca sexta, respectively, indicating that subunit E is shielded in V₁.