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Báo cáo khoa học: The transmembrane domain of subunitbof theEscherichia coli F1FOATP synthase is sufficient for H + -translocating activity together with subunitsaandc

Subunitbis indispensable for the formation of a functional H + -translocating FOcomplex both in vivo andin vitro. Whereas the very C-terminus of subunitbinteracts with F1 and plays a crucial role in enzyme assembly, the C-terminal region is also considered to be necessary for proper recon-stitution of FOinto liposomes. Here, we show that a syn-thetic peptide, residues 1–34 of subunitb(b1)34 ) [Dmitriev, O., Jones, P.C., Jiang,W. & Fillingame, R.H. (1999)J. Biol. Chem.274, 15598–15604], corresponding to the membrane domain of subunitbwas sufficient in forming an active FO complex when coreconstitutedwith purifiedacsubcomplex | Eur. J. Biochem. 271 3036-3042 2004 FEBS 2004 doi 10.1111 j.1432-1033.2004.04235.x The transmembrane domain of subunit b of the Escherichia coli F1FO ATP synthase is sufficient for H -translocating activity together with subunits a and c Jorg-Christian Greie Thomas Heitkamp and Karlheinz Altendorf Universitat OsnabrUck Fachbereich BiologiejChemie Abteilung Mikrobiologie OsnabrUck Germany Subunit b is indispensable for the formation of a functional H -translocating FO complex both in vivo and in vitro. Whereas the very C-terminus of subunit b interacts with F1 and plays a crucial role in enzyme assembly the C-terminal region is also considered to be necessary for proper reconstitution of FO into liposomes. Here we show that a synthetic peptide residues 1-34 of subunit b b1 34 Dmitriev O. Jones P.C. Jiang W. Fillingame R.H. 1999 J. Biol. Chem. 274 15598-15604 corresponding to the membrane domain of subunit b was sufficient in forming an active FO complex when coreconstituted with purified ac subcomplex. H translocation was shown to be sensitive to the specific inhibitor N N -dicyclohexylcarbodiimide and the resulting FO complexes were deficient in binding of isolated F1. This demonstrates that only the membrane part of subunit b is sufficient as well as necessary for H translocation across the membrane whereas the binding of F1 to FO is mainly triggered by C-terminal residues beyond Glu34 in subunit b. Comparison of the data with former reconstitution experiments additionally indicated that parts of the hydrophilic portion of the subunit b dimer are not involved in the process of ion translocation itself but might organize subunits a and c in FO assembly. Furthermore the data obtained functionally support the monomeric NMR structure of the synthetic b _3.1. Keywords F1FO ATP synthase subunit b reconstitution proton translocation Escherichia coli. Membrane-bound F-type ATPases F1FO occur ubiquitously in mitochondria chloroplasts and Bacteria. They reversibly catalyze