Labeling of individual amino acid residues in the membrane-embedded F0 part of the F1 F0 ATP synthase from Neurospora crassa. Influence of oligomycin and dicyclohexylcarbodiimide.
Three F0 subunits and the F1 subunit beta of the ATP synthase from Neurospora crassa were labeled with the lipophilic photoactivatable reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID). In the proteolipid subunit which was the most heavily labeled polypeptide labeling was confined to five residues at the NH2-terminus and five residues at the C-terminus of the protein. Labeling occurred at similar positions compared with the homologous protein (subunit c) in the ATP synthase from Escherichia coli, indicating a similar structure of the proteolipid subunits in their respective organisms. The inhibitors oligomycin and dicyclohexylcarbodiimide did not change the pattern of accessible surface residues in the proteolipid, suggesting that neither inhibitor induces gross conformational changes. However, in the presence of oligomycin, the extent of labeling in some residues was reduced. Apparently, these residues provide part of the binding site for the inhibitor. After reaction with dicyclohexylcarbodiimide an additional labeled amino acid was found at position 65 corresponding to the invariant carbodiimide-binding glutamic acid. These results and previous observations indicate that the carboxyl side chain of Glu-65 is located at the protein-lipid interphase. The idea is discussed that proton translocation occurs at the interphase between different types if F0 subunits. Dicyclohexylcarbodiimide or oligomycin might disturb this essential interaction between the F0 subunits.
Hoppe
J
J
Gatti
D
D
Weber
H
H
Sebald
W
W
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis*; D001389:Azirines; D001665:Binding Sites; D002234:Carbodiimides / Q000494:pharmacology*; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology*; D007457:Iodine Radioisotopes; D008928:Mitochondria / Q000201:enzymology; D009491:Neurospora / Q000201:enzymology*; D009492:Neurospora crassa / Q000201:enzymology*; D009840:Oligomycins / Q000494:pharmacology*; D011510:Proteolipids / Q000032:analysis; D006180:Proton-Translocating ATPases / Q000032:analysis*; D011522:Protons
D000596:Amino Acids; D001389:Azirines; D002234:Carbodiimides; D007457:Iodine Radioisotopes; D009840:Oligomycins; D011510:Proteolipids; D011522:Protons; D004024:Dicyclohexylcarbodiimide; C033174:3-(trifluoromethyl)-3-(3-iodophenyl)diazirine; D006180:Proton-Translocating ATPases