Membrane topography of the coupling ion binding site in Na+-translocating F1F0 ATP synthase.
A carbodiimide with a photoactivatable diazirine substituent was synthesized and incubated with the Na(+)-translocating F(1)F(0) ATP synthase from both Propionigenium modestum and Ilyobacter tartaricus. This caused severe inhibition of ATP hydrolysis activity in the absence of Na(+) ions but not in its presence, indicating the specific reaction with the Na(+) binding c-Glu(65) residue. Photocross-linking was investigated with the substituted ATP synthase from both bacteria in reconstituted 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC)-containing proteoliposomes. A subunit c/POPC conjugate was found in the illuminated samples but no a-c cross-links were observed, not even after ATP-induced rotation of the c-ring. Our substituted diazirine moiety on c-Glu(65) was therefore in close contact with phospholipid but does not contact subunit a. Na(+)in/(22)Na(+)out exchange activity of the ATP synthase was not affected by modifying the c-Glu(65) sites with the carbodiimide, but upon photoinduced cross-linking, this activity was abolished. Cross-linking the rotor to lipids apparently arrested rotational mobility required for moving Na(+) ions back and forth across the membrane. The site of cross-linking was analyzed by digestions of the substituted POPC using phospholipases C and A(2) and by mass spectroscopy. The substitutions were found exclusively at the fatty acid side chains, which indicates that c-Glu(65) is located within the core of the membrane.
von Ballmoos
Christoph
C
Institut für Mikrobiologie der Eidgenössischen Technischen Hochschule, ETH Zentrum, CH-8092 Zürich, Switzerland.
Appoldt
Yvonne
Y
Brunner
Josef
J
Granier
Thierry
T
Vasella
Andrea
A
Dimroth
Peter
P
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000302:isolation & purification; D001665:Binding Sites; D002234:Carbodiimides / Q000138:chemical synthesis; D027682:Cation Transport Proteins / Q000737:chemistry* / Q000302:isolation & purification; D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure; D005673:Fusobacterium / Q000201:enzymology; D007477:Ions / Q000378:metabolism; D008081:Liposomes; D008968:Molecular Conformation; D010713:Phosphatidylcholines; D021122:Protein Subunits; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000302:isolation & purification; D012964:Sodium / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D002234:Carbodiimides; D027682:Cation Transport Proteins; D007477:Ions; D008081:Liposomes; D010713:Phosphatidylcholines; D021122:Protein Subunits; D012964:Sodium; D000251:Adenosine Triphosphatases; C021838:sodium-translocating ATPase; D006180:Proton-Translocating ATPases; C028694:1-palmitoyl-2-oleoylphosphatidylcholine