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Organization of Subunits in the Membrane Domain of the Bovine F-ATPase Revealed by Covalent Cross-linking.

Lee J, Ding S, Walpole TB, Holding AN, Montgomery MG, Fearnley IM, Walker JE - J. Biol. Chem. (2015)

Bottom Line: The membrane domain contains six additional subunits named ATP8, e, f, g, DAPIT (diabetes-associated protein in insulin-sensitive tissues), and 6.8PL (6.8-kDa proteolipid), each with a single predicted transmembrane α-helix, but their orientation and topography are unknown.Cross-links involving the supernumerary subunits, where the structures are not known, show that the C terminus of ATP8 extends ∼70 Å from the membrane into the peripheral stalk and that the N termini of the other supernumerary subunits are on the same side of the membrane, probably in the mitochondrial matrix.These experiments contribute significantly toward building up a complete structural picture of the F-ATPase.

View Article: PubMed Central - PubMed

Affiliation: From the The Medical Research Council Mitochondrial Biology Unit, Cambridge Biomedical Campus, Hills Road, Cambridge CB2 0XY, United Kingdom and.

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Effect of varying the reaction conditions on the cross-linking of the bovine F-ATPase. The enzyme (1.16 mg/ml) was reacted at 37 °C with DSS(d0/d12) (lanes a–f) or BS3(d0/d12), DSG(d0/d6), or DSSG(d0/d6), (lanes g–i, respectively) at final reagent concentrations of 2 mm. For reference, the molecular weights of subunits α, oscp, and ϵ are 55,200, 20,900, and 5,600, respectively. The samples of modified enzyme were analyzed by SDS-PAGE (lanes a–i) and by blue native PAGE (lanes j and k). M denotes unreacted enzyme; lanes a–c, enzyme reacted with DSS(d0/d12) for 2, 4, and 20 h; lanes d–f, enzyme reacted with DSS(d0/d12) for 2 h at pH values of 8.0, 7.3, and 6.8; lanes g–i, enzyme reacted for 2 h at pH 8.0 with BS3(d0/d12), DSG(d0/d6), or DSSG(d0/d6), respectively; lane j, enzyme reacted with DSS(d0/d12) for 2 h at pH 8.0; lane k, bovine inner mitochrondrial membranes solubilized in the presence of n-dodecyl-β-d-maltose-neopentyl glycol. The migration positions of unmodified subunits of the enzyme are indicated on the left and on the right of native respiratory complexes I, III, and IV (CI, CIII, and CIV, respectively).
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Figure 2: Effect of varying the reaction conditions on the cross-linking of the bovine F-ATPase. The enzyme (1.16 mg/ml) was reacted at 37 °C with DSS(d0/d12) (lanes a–f) or BS3(d0/d12), DSG(d0/d6), or DSSG(d0/d6), (lanes g–i, respectively) at final reagent concentrations of 2 mm. For reference, the molecular weights of subunits α, oscp, and ϵ are 55,200, 20,900, and 5,600, respectively. The samples of modified enzyme were analyzed by SDS-PAGE (lanes a–i) and by blue native PAGE (lanes j and k). M denotes unreacted enzyme; lanes a–c, enzyme reacted with DSS(d0/d12) for 2, 4, and 20 h; lanes d–f, enzyme reacted with DSS(d0/d12) for 2 h at pH values of 8.0, 7.3, and 6.8; lanes g–i, enzyme reacted for 2 h at pH 8.0 with BS3(d0/d12), DSG(d0/d6), or DSSG(d0/d6), respectively; lane j, enzyme reacted with DSS(d0/d12) for 2 h at pH 8.0; lane k, bovine inner mitochrondrial membranes solubilized in the presence of n-dodecyl-β-d-maltose-neopentyl glycol. The migration positions of unmodified subunits of the enzyme are indicated on the left and on the right of native respiratory complexes I, III, and IV (CI, CIII, and CIV, respectively).

Mentions: The concentrations of cross-linkers, and the pH, duration, and temperature of reaction were optimized by varying the conditions and by monitoring the effect of cross-linking on the F-ATPase by SDS-PAGE and blue native-PAGE. The effect of varying the duration and pH of the reaction of DSS(d0/d12) with the enzyme is illustrated in Fig. 2 (lanes a–c and d–f, respectively). The effects of reaction of the enzyme with BS3(d0/d12), DSG(d0/d6), and DSSG(d0/d6) for 2 h at pH 8.0 and 37 °C are shown in lanes g–i in Fig. 2. The formation of intersubunit cross-links was indicated by the disappearance from the SDS-PAGE gels of the monomeric subunits of the enzyme accompanied by the formation of high molecular weight bands. The optimal conditions were considered to be those where the extent of formation of cross-linked products detected by SDS-PAGE analysis was greatest and the formation of multimeric cross-linked F-ATPase complexes, as detected by blue native-PAGE analysis, was minimal (Fig. 2, lane j). Those conditions are 2 μm F-ATPase (1.16 mg/ml) with 2 mm DSS (d0/d12) at pH 8.0 for 2 h at 37 °C. In lanes a–c, e, f, and h in Fig. 2, it is evident that the a- and c-subunits were unreactive with DSS(d0/d12), and in lane j, that cross-linking of the enzyme reduced its apparent molecular mass on blue native PAGE gels relative to unreacted enzyme (Fig. 2, lane k), presumably because the cross-links make the enzyme more compact.


Organization of Subunits in the Membrane Domain of the Bovine F-ATPase Revealed by Covalent Cross-linking.

Lee J, Ding S, Walpole TB, Holding AN, Montgomery MG, Fearnley IM, Walker JE - J. Biol. Chem. (2015)

Effect of varying the reaction conditions on the cross-linking of the bovine F-ATPase. The enzyme (1.16 mg/ml) was reacted at 37 °C with DSS(d0/d12) (lanes a–f) or BS3(d0/d12), DSG(d0/d6), or DSSG(d0/d6), (lanes g–i, respectively) at final reagent concentrations of 2 mm. For reference, the molecular weights of subunits α, oscp, and ϵ are 55,200, 20,900, and 5,600, respectively. The samples of modified enzyme were analyzed by SDS-PAGE (lanes a–i) and by blue native PAGE (lanes j and k). M denotes unreacted enzyme; lanes a–c, enzyme reacted with DSS(d0/d12) for 2, 4, and 20 h; lanes d–f, enzyme reacted with DSS(d0/d12) for 2 h at pH values of 8.0, 7.3, and 6.8; lanes g–i, enzyme reacted for 2 h at pH 8.0 with BS3(d0/d12), DSG(d0/d6), or DSSG(d0/d6), respectively; lane j, enzyme reacted with DSS(d0/d12) for 2 h at pH 8.0; lane k, bovine inner mitochrondrial membranes solubilized in the presence of n-dodecyl-β-d-maltose-neopentyl glycol. The migration positions of unmodified subunits of the enzyme are indicated on the left and on the right of native respiratory complexes I, III, and IV (CI, CIII, and CIV, respectively).
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Related In: Results  -  Collection

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Figure 2: Effect of varying the reaction conditions on the cross-linking of the bovine F-ATPase. The enzyme (1.16 mg/ml) was reacted at 37 °C with DSS(d0/d12) (lanes a–f) or BS3(d0/d12), DSG(d0/d6), or DSSG(d0/d6), (lanes g–i, respectively) at final reagent concentrations of 2 mm. For reference, the molecular weights of subunits α, oscp, and ϵ are 55,200, 20,900, and 5,600, respectively. The samples of modified enzyme were analyzed by SDS-PAGE (lanes a–i) and by blue native PAGE (lanes j and k). M denotes unreacted enzyme; lanes a–c, enzyme reacted with DSS(d0/d12) for 2, 4, and 20 h; lanes d–f, enzyme reacted with DSS(d0/d12) for 2 h at pH values of 8.0, 7.3, and 6.8; lanes g–i, enzyme reacted for 2 h at pH 8.0 with BS3(d0/d12), DSG(d0/d6), or DSSG(d0/d6), respectively; lane j, enzyme reacted with DSS(d0/d12) for 2 h at pH 8.0; lane k, bovine inner mitochrondrial membranes solubilized in the presence of n-dodecyl-β-d-maltose-neopentyl glycol. The migration positions of unmodified subunits of the enzyme are indicated on the left and on the right of native respiratory complexes I, III, and IV (CI, CIII, and CIV, respectively).
Mentions: The concentrations of cross-linkers, and the pH, duration, and temperature of reaction were optimized by varying the conditions and by monitoring the effect of cross-linking on the F-ATPase by SDS-PAGE and blue native-PAGE. The effect of varying the duration and pH of the reaction of DSS(d0/d12) with the enzyme is illustrated in Fig. 2 (lanes a–c and d–f, respectively). The effects of reaction of the enzyme with BS3(d0/d12), DSG(d0/d6), and DSSG(d0/d6) for 2 h at pH 8.0 and 37 °C are shown in lanes g–i in Fig. 2. The formation of intersubunit cross-links was indicated by the disappearance from the SDS-PAGE gels of the monomeric subunits of the enzyme accompanied by the formation of high molecular weight bands. The optimal conditions were considered to be those where the extent of formation of cross-linked products detected by SDS-PAGE analysis was greatest and the formation of multimeric cross-linked F-ATPase complexes, as detected by blue native-PAGE analysis, was minimal (Fig. 2, lane j). Those conditions are 2 μm F-ATPase (1.16 mg/ml) with 2 mm DSS (d0/d12) at pH 8.0 for 2 h at 37 °C. In lanes a–c, e, f, and h in Fig. 2, it is evident that the a- and c-subunits were unreactive with DSS(d0/d12), and in lane j, that cross-linking of the enzyme reduced its apparent molecular mass on blue native PAGE gels relative to unreacted enzyme (Fig. 2, lane k), presumably because the cross-links make the enzyme more compact.

Bottom Line: The membrane domain contains six additional subunits named ATP8, e, f, g, DAPIT (diabetes-associated protein in insulin-sensitive tissues), and 6.8PL (6.8-kDa proteolipid), each with a single predicted transmembrane α-helix, but their orientation and topography are unknown.Cross-links involving the supernumerary subunits, where the structures are not known, show that the C terminus of ATP8 extends ∼70 Å from the membrane into the peripheral stalk and that the N termini of the other supernumerary subunits are on the same side of the membrane, probably in the mitochondrial matrix.These experiments contribute significantly toward building up a complete structural picture of the F-ATPase.

View Article: PubMed Central - PubMed

Affiliation: From the The Medical Research Council Mitochondrial Biology Unit, Cambridge Biomedical Campus, Hills Road, Cambridge CB2 0XY, United Kingdom and.

Show MeSH