Topological studies suggest that the pathway of the protons through F0 is provided by amino acid residues accessible from the lipid phase

Abstract

Summary -The structure of the F 0 part of ATP synthases from E. coli and Neurospora crassa was analyzed by hydrophobic surface labeling with [ 125 I]TID. In the E. co/i F 0 all three subunits were freely accessible to the reagent, suggesting that these subunits are independently integrated in the membrane. Labeted amino acid residues were identified by Edman degradation of the dicyclohexylcarbodiimide binding (DCCD) proteins from E. coli and Neurospora crassa. The very similar patterns obtained with the two homologaus proteins suggested the existence of tightly packed cx-helices. The oligomeric structure of the DCCD binding protein appeared to be very rigid since little, if any, change in the labeling patternwas observed upon addition of oligomycin or DCCD to membranes from Neurospora crassa. When membrancs were pretrcated with DCCD prior to the reaction with [1 25 I]TID an additionally labeled amino acid appeared at the position of Glu·65 which binds DCCD covalently, indicating the Jocation of this inhibitor on the outside of the oligomer. It is suggested that proton conduction occurs at the surface of the oligomer of the DCCD binding protein. Possibly this oligomer rotates against the subunit a or b and thus enables proton translocation. Conserved residues in subunit a, probably located in the Iipid bilayer, might participate in the pro· ton translocation mechanism.

Amino acid substitutions in subunit 9 of the mitochondrial ATPase complex of Saccharomyces cerevisiae

“…2), has led us to suggest that the membraneembedded portion of the N-terminal stem of subunit 9 extends from IlelO to Ile34 and that of the C-terminal stem extends from Ala51 to Gly75. These correspond within one or two amino acids to those suggested by Hoppe and Sebald [20] for the relevant domains of subunit c of the E. coli protontranslocating ATPase complex. By contrast, Senior and Wise [21] propose that the exposed loop region is 2 or 3 amino acids shorter at each end, so that the first exposed residue at the N-terminal edge of the loop is considered to be the absolutely conserved arginine residue (position 41 in E. coli and 39 in yeast).…”

“…This independence may occur because the trans-membrane domains of subunit 9 which are involved in oligomycin binding [4-91 participate directly in the activity of proton translocation (cf. [20,26]), whereas the loop region may have a distinct role in energy-coupling between the Fo and F1 sectors (see above).…”

Amino acid substitutions in mitochondrial ATPase subunit 6 of Saccharomyces cerevisiae leading to oligomycin resistance

“…The altered subunit 6 structure in the oliR mutants could then allosterically alter the oligomycin binding site of subunit 9 (cf. [5,7]). This could be mediated, for example, by direct interaction of the transmembrane domains represented in fig.2.…”

“…This could be mediated, for example, by direct interaction of the transmembrane domains represented in fig.2. Current concepts of the topological organization [4,5] of the Fo-sector of the E. coli protontranslocating ATPase complex place emphasis on dynamic structural and functional interactions be-82 SUBUNIT 6 SUBUNIT 9 I I I I Fig.2. Location of amino acid substitutions conferring oligomycin resistance in membrane-spanning domains of mtATPase subunit 6 and 9.…”

“…Subunit 6 has been implicated [2] in the coupling of proton translocation to ATP synthesis catalyzed by the soluble Ft-sector of the complex. The translocation of protons is DCCD, dicyclohexyl carbodiimide; mtATPase, mitochondrial proton-translocating ATPase; oliR, oligomycin-resistant (in reference to phenotype) generally considered [3] to be primarily effected by subunit 9, the 76 amino acid DCCD-binding proteolipid; it has further been suggested [4,5] that subunit 6 may itself play a role in this process.…”