title: Expression, purification, and characterization of subunit E, an essential subunit of the vacuolar ATPase.
abstract: 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 (TF(1)). The alpha(3)beta(3)E-hybrid-complex had 56% of the ATPase activity of the native TF(1). By comparison, an alpha(3)beta(3)-formation without Vma4p showed about 24% of total TF(1) 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).
authors.affiliation: Fachrichtung 2.5-Biophysik, Universität des Saarlandes, D-66421 Homburg, Germany. ggrueber@med-rz.uni-saarland.de
authors.forename: Gerhard
authors.initials: G
authors.lastname: Grüber
authors.forename: Jasminka
authors.initials: J
authors.lastname: Godovac-Zimmermann
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Link
authors.forename: Unal
authors.initials: U
authors.lastname: Coskun
authors.forename: Vincenzo F
authors.initials: VF
authors.lastname: Rizzo
authors.forename: Christian
authors.initials: C
authors.lastname: Betz
authors.forename: Susanne M
authors.initials: SM
authors.lastname: Bailer
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002942:Circular Dichroism; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D018402:Manduca / Q000201:enzymology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000378:metabolism; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry*
chemical_list: D017931:DNA Primers; D010446:Peptide Fragments; D014357:Trypsin; D025262:Vacuolar Proton-Translocating ATPases