Arrangement of the Multicopy H+-translocating Subunit c in the Membrane Sector of the Escherichia coliF1F0 ATP Synthase*

The multicopy subunit c of the H+-transporting F1F0 ATP synthase of Escherichia coli is thought to fold across the membrane as a hairpin of two hydrophobic α-helices. The conserved Asp61, centered in the second transmembrane helix, is essential for H+ transport. In this study, we have made sequential Cys substitutions across both transmembrane helices and used disulfide cross-link formation to determine the oligomeric arrangement of the c subunits. Cross-link formation between single Cys substitutions in helix 1 provided initial limitations on how the subunits could be arranged. Double Cys substitutions at positions 14/16, 16/18, and 21/23 in helix 1 and 70/72 in helix 2 led to the formation of cross-linked multimers upon oxidation. Double Cys substitutions in helix 1 and helix 2, at residues 14/72, 21/65, and 20/66, respectively, also formed cross-linked multimers. These results indicate that at least 10 and probably 12 subunits c interact in a front-to-back fashion to form a ring-like arrangement in F0. Helix 1 packs at the interior and helix 2 at the periphery of the ring. The model indicates that the Asp61carboxylate is centered between the helical faces of adjacent subunit c at the center of a four-helix bundle.

[1]  P. Dimroth,et al.  Primary sodium ion translocating enzymes. , 1997, Biochimica et biophysica acta.

[2]  S. Vik,et al.  A mechanism of proton translocation by F1F0 ATP synthases suggested by double mutants of the a subunit. , 1994, The Journal of biological chemistry.

[3]  W. Junge,et al.  Intersubunit rotation in active F-ATPase , 1996, Nature.

[4]  S. Schuldiner,et al.  Reserpine as a competitive and reversible inhibitor of the catecholamine transporter of bovine chromaffin granules , 1979, FEBS letters.

[5]  G. Feher,et al.  The bacterial photosynthetic reaction center as a model for membrane proteins. , 1989, Annual review of biochemistry.

[6]  R. H. Fillingame,et al.  The essential carboxyl group in subunit c of the F1F0 ATP synthase can be moved and H(+)-translocating function retained. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[7]  C. Tang,et al.  The Stalk Region of the Escherichia coli ATP Synthase , 1996, The Journal of Biological Chemistry.

[8]  S. Rahlfs,et al.  Sequence of subunit c of the Na+‐translocating F1FO ATPase of Acetobacterium woodii: proposal for determinants of Na+ specificity as revealed by sequence comparisons , 1997, FEBS letters.

[9]  P. Boyer.  The ATP synthase--a splendid molecular machine. , 1997, Annual review of biochemistry.

[10]  R. H. Fillingame,et al.  Stoichiometry of subunits in the H+-ATPase complex of Escherichia coli. , 1982, The Journal of biological chemistry.

[11]  W. Junge,et al.  ATP synthase: a tentative structural model , 1997, FEBS letters.

[12]  H. Wieczorek,et al.  Animal plasma membrane energization by chemiosmotic H+ V-ATPases. , 1997, The Journal of experimental biology.

[13]  Jan Pieter Abrahams,et al.  Structure at 2.8 Â resolution of F1-ATPase from bovine heart mitochondria , 1994, Nature.

[14]  Jeffrey H. Miller,et al.  A short course in bacterial genetics , 1992 .

[15]  P. Dimroth,et al.  Kinetics of inactivation of the F1Fo ATPase of Propionigenium modestum by dicyclohexylcarbodiimide in relationship to H+ and Na+ concentration: probing the binding site for the coupling ions. , 1993, Biochemistry.

[16]  R Henderson,et al.  Electron-crystallographic refinement of the structure of bacteriorhodopsin. , 1996, Journal of molecular biology.

[17]  H. Schägger,et al.  Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. , 1987, Analytical biochemistry.

[18]  M. Girvin,et al.  Determination of local protein structure by spin label difference 2D NMR: the region neighboring Asp61 of subunit c of the F1F0 ATP synthase. , 1994, Biochemistry.

[19]  T. Stevens,et al.  Structure, function and regulation of the vacuolar (H+)-ATPase. , 1997, Annual review of cell and developmental biology.

[20]  I. Paulsen,et al.  Phylogenetic analyses of the homologous transmembrane channel-forming proteins of the F0F1-ATPases of bacteria, chloroplasts and mitochondria. , 1996, Microbiology.

[21]  S. Howitt,et al.  The coupling of the relative movement of thea andc subunits of the F0 to the conformational changes in the F1-ATPase , 1996, Journal of bioenergetics and biomembranes.

[22]  R. Aggeler,et al.  Cross-linking of the δ Subunit to One of the Three α Subunits Has No Effect on Functioning, as Expected if δ Is a Part of the Stator That Links the F1 and F0 Parts of the Escherichia coli ATP Synthase* , 1997, The Journal of Biological Chemistry.

[23]  The F0 complex of the Escherichia coli ATP synthase. Investigation by electron spectroscopic imaging and immunoelectron microscopy. , 1995, European journal of biochemistry.

[24]  J. Brunner,et al.  Structure of the membrane-embedded F0 part of F1F0 ATP synthase from Escherichia coli as inferred from labeling with 3-(Trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine. , 1984, Biochemistry.

[25]  John B. C. Findlay,et al.  The first putative transmembrane helix of the 16 kDa proteolipid lines a pore in the Vo sector of the vacuolar H(+)-ATPase. , 1995, Biochemical Journal.

[26]  N. W. Isaacs,et al.  Crystal structure of an integral membrane light-harvesting complex from photosynthetic bacteria , 1995, Nature.

[27]  J. Walker,et al.  The unc operon. Nucleotide sequence, regulation and structure of ATP-synthase. , 1984, Biochimica et biophysica acta.

[28]  George Oster,et al.  Energy transduction in ATP synthase , 1998, Nature.

[29]  H. Towbin,et al.  Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[30]  Kazuhiko Kinosita,et al.  Direct observation of the rotation of F1-ATPase , 1997, Nature.

[31]  M. Mandel,et al.  cDNA sequence encoding the 16-kDa proteolipid of chromaffin granules implies gene duplication in the evolution of H+-ATPases. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[32]  I. Yamato,et al.  A gene encoding the 16-kDa proteolipid subunit of Enterococcus hirae Na(+)-ATPase complex. , 1993, Biochemical and biophysical research communications.

[33]  R. H. Fillingame,et al.  Assembly of F0 Sector of Escherichia coli H+ ATP Synthase , 1995, The Journal of Biological Chemistry.

[34]  A. E. Senior,et al.  ATP synthesis by oxidative phosphorylation. , 1988, Physiological reviews.

[35]  V. V. Bulygin,et al.  Rotation of subunits during catalysis by Escherichia coli F1-ATPase. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[36]  G. Grüber,et al.  Structural changes in the γ and ε subunits of theEscherichia coli F1F0-type ATPase during energy coupling , 1996 .

[37]  R. H. Fillingame,et al.  Coupling H+ transport and ATP synthesis in F1F0-ATP synthases: glimpses of interacting parts in a dynamic molecular machine. , 1997, The Journal of experimental biology.

[38]  M. Girvin,et al.  Organization of the F0 sector of Escherichia coli H+-ATPase: the polar loop region of subunit c extends from the cytoplasmic face of the membrane. , 1989, Biochemistry.

[39]  D. Keller,et al.  Topographical structure of membrane‐bound Escherichia coli F1F0 ATP synthase in aqueous buffer , 1996, FEBS letters.

[40]  R. L. Cross,et al.  Gene duplication as a means for altering H+/ATP ratios during the evolution of Fo F1 ATPases and synthases , 1990, FEBS letters.

[41]  Y. Zhang,et al.  Subunits Coupling H+ Transport and ATP Synthesis in the Escherichia coli ATP Synthase , 1995, The Journal of Biological Chemistry.

[42]  U. Hahn,et al.  A general method for rapid site-directed mutagenesis using the polymerase chain reaction. , 1990, Gene.

[43]  E. Eliopoulos,et al.  Structure of a 16 kDa integral membrane protein that has identity to the putative proton channel of the vacuolar H(+)-ATPase. , 1992, Protein engineering.

[44]  K Takeyasu,et al.  Molecular imaging of Escherichia coli F0F1‐ATPase in reconstituted membranes using atomic force microscopy , 1996, FEBS letters.

[45]  U. Gerike,et al.  Molecular basis for the coupling ion selectivity of F1F0 ATP synthases: probing the liganding groups for Na+ and Li+ in the c subunit of the ATP synthase from Propionigenium modestum. , 1997, Biochemistry.

[46]  W. Junge,et al.  Cross-linking of Engineered Subunit δ to (αβ)3 in Chloroplast F-ATPase* , 1996, The Journal of Biological Chemistry.

[47]  J. Findlay,et al.  Evidence for a common structure for a class of membrane channels. , 1993, European journal of biochemistry.

[48]  D. Jans,et al.  Hypothesis the mecanism of ATP synthase conformational change by rotation of the b-subunit , 1984 .

[49]  Y. Zhang,et al.  Changing the Ion Binding Specificity of the Escherichia coli H-transporting ATP Synthase by Directed Mutagenesis of Subunit c(*) , 1995, The Journal of Biological Chemistry.

[50]  R. H. Fillingame,et al.  Conserved polar loop region of Escherichia coli subunit c of the F1F0 H+-ATPase. Glutamine 42 is not absolutely essential, but substitutions alter binding and coupling of F1 to F0. , 1989, The Journal of biological chemistry.