ATP synthase complex from bovine heart mitochondria. Passive H+ conduction through F0 does not require oligomycin sensitivity-conferring protein.

Pringle, Michael J.; Kenneally, Mary; Joshi, Saroj

doi:10.1016/s0021-9258(19)39161-6

Cited by 19 publications

(13 citation statements)

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“…All standard DNA manipulations were carried out according to protocols described elsewhere (Joshi et al, 1996). Bovine heart mitochondria (Joshi & Sanadi, 1979), OSCP-depleted submitochondrial particles (AE-P) at pH 9.8 (Fessenden & Racker, 1967), purified F o fractions depleted of OSCP (UF o ) (Pringle et al, 1990), and soluble F 1 -ATPase (Horstman & Racker, 1970) were isolated as described previously. SDS-polyacrylamide gel eletrophoresis, electrotransfer, gel staining, Western blotting, and ELISA assays were carried out according to previously published protocols (Joshi & Burrows, 1990).…”

Section: Methodsmentioning

confidence: 99%

“…Reconstitution of Oligomycin-SensitiVe ATPase and [ 32 P]P i -ATP Exchange. Oligomycin-sensitive ATPase complexes were reconstituted by adding soluble F 1 and OSCP to fractions of ATP synthase that had been depleted previously of F 1 and OSCP (UF o ) as described in Pringle et al (1990).…”

Section: Methodsmentioning

confidence: 99%

“…After 30 min, 50 µL of 20 mM Tris‚HCl buffer, pH 8.5, was added. The ATPase activity was measured using a 10 µL aliquot (Pringle et al, 1990).…”

Section: Methodsmentioning

confidence: 99%

“…The oligomycin sensitivity conferring protein (OSCP), a major constituent of the stalk segment of mitochondrial ATP synthase, is presumably located in a "pit" next to a protrusion at the base of the F 1 particle (Abrahams et al, 1993;Kagawa & Racker, 1966;McLennan & Tzagoloff, 1968;Van de Stadt et al, 1972;Dupuis et al, 1985). Depletion/reconstitution studies of the ATP synthase show that OSCP can form independent complexes with both F 1 and F o segments, although it is required neither for F 1 -catalyzed inhibitorinsensitive ATP hydrolysis nor for F o -mediated passive proton translocation (Dupuis et al, 1985;Dupuis & Vignais, 1987;Pringle et al, 1990). It is also clear that OSCP is not obligatory for binding of F 1 to F o although it is absolutely essential for reassembly of oligomycin-and DCCD-sensitive ATPase and ATP synthesis activities in OSCP-depleted F 1 F o complexes (Kagawa & Racker, 1966;McLennan & Tzagoloff, 1968;Senior, 1971;Pringle et al, 1990;Joshi & Huang, 1991).…”

mentioning

confidence: 99%

“…Depletion/reconstitution studies of the ATP synthase show that OSCP can form independent complexes with both F 1 and F o segments, although it is required neither for F 1 -catalyzed inhibitorinsensitive ATP hydrolysis nor for F o -mediated passive proton translocation (Dupuis et al, 1985;Dupuis & Vignais, 1987;Pringle et al, 1990). It is also clear that OSCP is not obligatory for binding of F 1 to F o although it is absolutely essential for reassembly of oligomycin-and DCCD-sensitive ATPase and ATP synthesis activities in OSCP-depleted F 1 F o complexes (Kagawa & Racker, 1966;McLennan & Tzagoloff, 1968;Senior, 1971;Pringle et al, 1990;Joshi & Huang, 1991). Thus, the primary role of OSCP is possibly in the transmission of the energy of the proton electrochemical gradient between F o and F 1 , thereby facilitating the "coupling" of an "uncoupled" F 1 -F o complex.…”

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confidence: 99%

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

et al. 1997

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Earlier studies on oligomycin sensitivity conferring protein (OSCP) of bovine mitochondrial ATP synthase (F1Fo) indicated that a deletion mutant form (CD-10), lacking the last 10 amino acid residues (K181-L190), was unable to bind to the Fo segment, or reconstitute energy-linked reactions in OSCP-depleted F1Fo complexes [Joshi et al. (1996) Biochemistry 35, 12094-12103]. So far as known, the K181-L190 region of all mammalian species of OSCP harbors four charged residues at positions 181, 184, 187, and 188, while secondary structure predictions suggest that the K178-M186 region has a high propensity to form a helix [Ovchinnikov et al. (1984) FEBS Lett. 166, 19-22; Higuti et al. (1993) Biochim. Biophys. Acta 1172, 311-314; Grinkevich et al. ( 1994) Biol. Membr. 11, 310-323; Engelbrecht et al. (1991) Z. Naturforsch., C: Biochem., Biophys., Biol.,Virol. 46, 759-764]. Present studies were undertaken to clarify the role of individual amino acids in the K181-L190 region in OSCP-stimulated energy coupling. Our data show that simultaneous replacements of all four charged residues by uncharged but polar glutamines, or of K181-R184 by apolar alanines, had no significant influence either on the total alpha-helix content of the mutant forms or on the ability of mutant OSCPs to couple energy-linked reactions. However, a substitution of the K181-M186 region by six proline residues led to complete loss in the coupling activity of the resultant mutant. A detailed analysis of the 6-proline mutant form revealed that the variant was indistinguishable from WT OSCP with respect to expression characteristics, affinity for S-Sepharose, and ability to interact with F1, but was unable to complex with the Fo segment. These studies suggest that the global protein structure was not destabilized. The helix potential prediction analyses showed that the 6-proline OSCP displayed a marked decrease in the helix-forming propensity in the region corresponding to residues 175-190. Quantitative CD analyses to measure helical content demonstrated that both of the mutant forms 6-proline-OSCP and CD-10 had a somewhat lower alpha-helical content compared to WT protein, while synthetic peptides corresponding in sequence to the K178-L190 region displayed a high propensity to form a helix. Taken together, these results suggest that the C-terminal end of OSCP encompasses an alpha-helix which is crucial for high-affinity interactions of the C-terminal end of this subunit with Fo in the F1Fo enzyme.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…After 30 min, 50 µL of 20 mM Tris‚HCl buffer, pH 8.5, was added. The ATPase activity was measured using a 10 µL aliquot (Pringle et al, 1990).…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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

et al. 1997

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ATP synthase complex from bovine heart mitochondria: The oligomycin sensitivity conferring protein is essential for dicyclohexyl carbodiimide-sensitive ATPase

Joshi

Huang

1991

Biochimica et Biophysica Acta (BBA) - Biomembranes

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The γ subunit of F₁ and the PVP protein of F_o (F_oI) are components of the gate of the mitochondrial F_oF₁ H⁺‐ATP synthase

Papa¹,

Guerrieri²,

Zanotti³

et al. 1990

FEBS Letters

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The γ subunit of the F1 moiety of the bovine mitochondrial H+‐ATP synthase is shown to function as a component of the gate. Addition of purified γ subunit to Fo‐liposomes inhibits transmembrane proton conduction. This inhibition can be removed by the bifunctional thiol reagent diamide. Immunoblot analysis shows that the diamide effect is likely due to disulphide bridging of the γ subunit with the PVP protein of the Fo sector.

show abstract

ATP synthase complex from bovine heart mitochondria. Passive H+ conduction through F0 does not require oligomycin sensitivity-conferring protein.

Cited by 19 publications

References 40 publications

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

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

ATP synthase complex from bovine heart mitochondria: The oligomycin sensitivity conferring protein is essential for dicyclohexyl carbodiimide-sensitive ATPase

The γ subunit of F₁ and the PVP protein of F_o (F_oI) are components of the gate of the mitochondrial F_oF₁ H⁺‐ATP synthase

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ATP synthase complex from bovine heart mitochondria. Passive H+ conduction through F0 does not require oligomycin sensitivity-conferring protein.

Cited by 19 publications

References 40 publications

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

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

ATP synthase complex from bovine heart mitochondria: The oligomycin sensitivity conferring protein is essential for dicyclohexyl carbodiimide-sensitive ATPase

The γ subunit of F1 and the PVP protein of Fo (FoI) are components of the gate of the mitochondrial FoF1 H+‐ATP synthase

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

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

The γ subunit of F₁ and the PVP protein of F_o (F_oI) are components of the gate of the mitochondrial F_oF₁ H⁺‐ATP synthase