Accessibility of F₀ subunits from Escherichia coli ATP synthase

Abstract

1. Antisera have been raised against denatured and non-denatured subunits a, b and c of the Fo complex of the ATP synthase from Escherichiu coli. The subunit specificity of the antibodies has been established with immunoblot analysis or enzyme-linked immunosorbent assay (ELISA).2. In inside-out oriented membrane vesicles the binding avidities of both sets of antisera, against denatured and non-denatured subunits of Fo, were similar in the presence as well as in the absence of the F1 part. F1-depleted everted membrane vesicles always produced an efficient binding of the different antisera. In the presence of F1 no antibody recognition could be observed with the anti-a antisera, while anti-b and anti-c antisera showed strong binding. However, a higher membrane protein concentration was necessary for the same antibody binding as in F1-stripped vesicles.3. In membrane vesicles with right-side-out orientation the recognition of the three Fo subunits was dependent on the antisera set used. Antisera raised against denatured subunits showed no binding to the membrane vesicles, only in case of anti-(dodecylsulfate-denatured b) antiserum could a slight affinity be detected. An antigen-antibody recognition with all three Fo subunits occurred when the antisera against non-denatured subunits were incubated with membrane vesicles of right-side-out orientation. The membrane protein concentration which was necessary to produce a significant binding was 10-100-fold higher compared to that of F,-depleted everted membrane vesicles.The ATP synthase (FIFO) of Escherichiu coli reversibly couples the synthesis or hydrolysis of ATP to the translocation of protons across the membrane. This enzyme consists of two structurally and functionally distinct entities: the watersoluble part (Fl), which carries the catalytic centers, and the membrane-integrated sector (Fo), which functions as a proton channel (for reviews, see [l -31). The Fo complex is composed of three different subunits a, b and c with relative molecular masses of 30276, 17265 and 8288, respectively and with the unusual stoichiometry of 1 : 2: 10 f 1 for a: b: c [4] (for reviews,Subunit c, also referred to as proteolipid or dicyclohexylcarbodiimide-binding protein, is the most extensively studied subunit of the Fo complex (for review, see [S] tyrosine residues [14] indicated that the hydrophilic turn is directed to the cytoplasmic side of the membrane. Antibody binding studies revealed that antigenic determinants of subunit c are accessible from the cytoplasm [15, 161. Subunit b has only a small hydrophobic segment at the N-terminal region with which it is possibly anchored into the membrane. The rest (80%) of the molecule is extremely hydrophilic [5, 61. This polar part is highly accessible to proteases and exposed to the cytoplasm Only a little is known about subunit a. Like subunit c, this protein is extremely hydrophobic spanning the membrane several times. The relative hydrophilic N-terminal region is probably exposed to the water phase [5, 61. Proteolytic degradation ...

Integration of b Subunits of Unequal Lengths into F1F0-ATP Synthase

“…Western blotting reagents and high performance chemiluminescence film were purchased from Amersham Biosciences. Polyclonal antibodies against SDS-denatured b subunit (35,36) were kindly provided by Dr. Karlheinz Altendorf (Universitä t Osnabrü ck, Osnabrü ck, Germany). Monoclonal antibodies against the V5 epitope were purchased from Invitrogen.…”

Deletions in the Second Stalk of F1F0-ATP Synthase in Escherichia coli

“…The anti-rabbit immunoglobulin horseradish peroxidase-linked whole antibody (from donkey), electrochemiluminescence Western blotting detection reagents, and Hyperfilm were obtained from Amersham Pharmacia Biotech. Polyclonal antibodies against SDS-denatured b subunit (26,27) were kindly provided by Dr. Karlheinz Altendorf (Universitä t Osnabrü ck, Osnabrü ck, Germany). Oligonucleotides were synthesized by the DNA synthesis core facility of the University of Florida Interdisciplinary Center for Biotechnology Research.…”

The F0 Complex of the Escherichia Coli ATP Synthase

“…The ATPase activity of F,-reconstituted proteoliposomes was measured according to Steffens et al (1984). SDS/polyacrylamide gel electrophoresis using gels containing 16% polyacrylamide and 6 M urea (Schagger and von Jagow, 1987) and immunoblotting (Deckers-Hebestreit and Altendorf, 1986;Hensel et al, 1990) were carried out as described.…”