TY - JOUR
T1 - Expression, purification, and characterization of subunit E, an essential subunit of the vacuolar ATPase
AU - Grüber, Gerhard
AU - Godovac-Zimmermann, Jasminka
AU - Link, Thomas A.
AU - Coskun, Ünal
AU - Rizzo, Vincenzo F.
AU - Betz, Christian
AU - Bailer, Susanne M.
PY - 2002
Y1 - 2002
N2 - 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.
AB - 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.
KW - Circular dichroism spectroscopy
KW - F ATPase
KW - Saccharomyces cerevisiae
KW - V ATPase
KW - Vacuolar ATPase
KW - Vma4p
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U2 - 10.1016/S0006-291X(02)02468-3
DO - 10.1016/S0006-291X(02)02468-3
M3 - Article
C2 - 12413952
AN - SCOPUS:0036430416
SN - 0006-291X
VL - 298
SP - 383
EP - 391
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
IS - 3
ER -