Topography of oligomycin sensitivity conferring protein in the mitochondrial adenosine triphosphatase-ATP synthase

Abstract

The topographical organization of oligomycin sensitivity conferring protein (OSCP) in the mitochondrial adenosinetriphosphatase (ATPase)-ATP synthase complex has been studied. The accessibility of OSCP to monoclonal antibodies has been qualitatively visualized by using the protein A-gold electron microscopy immunocytochemistry or quantitatively estimated by immunotitration of OSCP in depolymerized or intact membranes. Besides, OSCP cannot be labeled by 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID) which selectively labels the hydrophobic core of membrane proteins. These observations demonstrate an external location of OSCP on the inner face of the inner mitochondrial membrane. The position of OSCP relative to other peptides of the complex has been analyzed by cross-linking experiments using either zero length N-(ethoxycarbonyl)-2-ethoxydihydroquinoline or 11-A span dimethyl suberimidate cross-linkers in the ATPase-ATP synthase complex. The OSCP cross-linked products were identified either by immunocharacterization with anti-alpha, anti-beta, or anti-OSCP monoclonal antibodies or by their molecular weight. OSCP was cross-linked with either the alpha- or beta-subunits of F1 or to a subunit of Mr 24 000. Other types of cross-linking were obtained by the labeling of OSCP with [cysteamine-35S]-N-succinimidyl 3-[[2-((2-nitro-4-azidophenyl)amino)ethyl]dithio]propionate ([35S]SNAP) and reconstitution of SNAP-OSCP with F1 in urea-treated submitochondrial particles. Under these conditions, OSCP is found to be adjacent to two other peptides of molecular weight close to 30 000. A comparison is made between the topology and the organization of the b-subunit of Escherichia coli and OSCP, suggesting an analogy between OSCP and the hydrophilic part of the b-subunit.

Bacterial adenosine 5'-triphosphate synthase (F1F0): purification and reconstitution of F0 complexes and biochemical and functional characterization of their subunits.

“…Furthermore, there is experimental evidence that a 24-kDa component of an Fo preparation from beef heart is in fact identical to subunit 6 (91). In addition, the 24-kDa protein seems to be adjacent to OSCP as has been inferred from cross-linking studies (9,205). The precise role of this subunit is still unknown; however, as discussed for E. coli, it might have a structural function.…”

“…Both proteins are required for the binding of F1 to the membrane sector and for rendering the F1 complex sensitive to inhibition by oligomycin and DCCD (157,210). Cross-linking experiments with the F1Fo complex from pig heart mitochondria revealed that OSCP is in close proximity to a and ,B of F1 (9). Surprisingly, Sandri et al (161) recently provided evidence for an Fl-binding site on Fo in the absence of F6 and OSCP; however, the ATPase activity of this complex was insensitive towards DCCD.…”

Docking the mitochondrial inhibitor protein IF1 to a membrane receptor different from the F1‐ATPase β subunit

“…Cross-linking of membrane bound IF, with N-(ethoxycarbonyl)-Zethoxy-l-2-dihydroquinoline (EEDQ) was performed as described previously for the study of nearest neighbours of oligomycin-sensitivity-conferring protein in the mitochondria1 membrane (Archinard et al, 1986). Cross-linking experiments with 0.5 mM bis(2-[succinimidooxycarbonyloxy]ethyl)sulfone) (BSOCOES) and ETP (0.9 mg/ml in 25 mM sodium phosphate, pH 7.5) were performed for 15 min at 0°C in the presence of 0.5 mM BSOCOES.…”

“…Cross-linking of membrane bound IF, with N-(ethoxycarbonyl)-Zethoxy-l-2-dihydroquinoline (EEDQ) was performed as described previously for the study of nearest neighbours of oligomycin-sensitivity-conferring protein in the mitochondria1 membrane (Archinard et al, 1986). Experimental conditions were as described in Materials and Methods.…”

Oligomycin Sensitivity Conferring Protein (OSCP) of Bovine Heart Mitochondrial ATP Synthase: High-Affinity OSCP−Fo Interactions Require a Local α-Helix at the C-Terminal End of the Subunit

“…Based on the data presented, we suggest that (i) the K181-L190 region at the C-terminal end of OSCP encompasses an R-helix or part of an R-helix, and (ii) interactions between the C-terminal end of OSCP and subunit(s) of F o in the F 1 -F o enzyme do not specifically require acidic or basic residues, or residues with uncharged side chain, but do involve an R-helix. F 1 F o , as well as cross-linking of intact F 1 F o enzyme using cross-linking reagents of different chemical reactivities and effective distances (0-12 Å) (Archinard et al, 1986;Joshi & Burrows, 1990;Belogrudov et al, 1995). Beckers et al (1992) showed formation of a cross-link between a basic amino acid residue of CF 1 δ and an acidic residue at the C-terminal end of CF o b.…”