Expression, purification, and characterization of subunit E, an essential subunit of the vacuolar ATPase

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% alpha-helix and 23% beta-sheet content. Vma4p formed a hybrid-complex with the nucleotide-binding subunits alpha and beta of the closely related F-1. ATPase of the thermophilic bacterium PS3 (TF1). The alpha(3)beta(3)E-hybrid-complex had 56% of the ATPase activity of the native TF1. By comparison, an alpha(3)beta(3)-formation without Vma4p showed about 24% of total TF1 ATPase activity. This is the first demonstration of a hydrolytically active hybrid-complex consisting of F-1 and V-1 subunits. The arrangement of subunit E in V-1 has been probed using the recombinant Vma4p, the alpha(3)beta(3)E-hybrid-complex together with V-1 and an A(3)B(3)HEG-subcomplex of the V-1 ATPase from Manduca sexta, respectively, indicating that subunit E is shielded in V-1. (C) 2002 Elsevier Science (USA). All rights reserved.