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Journal article

Membrane topography of the coupling ion binding site in Na+-translocating F1F0 ATP synthase.

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  • 2001-11-24
Published in:
  • The Journal of biological chemistry. - 2002
Adenosine Triphosphatases
Binding Sites
Carbodiimides
Cation Transport Proteins
Cell Membrane
Fusobacterium
Ions
Liposomes
Molecular Conformation
Phosphatidylcholines
Protein Subunits
Proton-Translocating ATPases
Sodium
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
English 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.
Language
  • English
Open access status
bronze
Identifiers
Persistent URL
https://sonar.rero.ch/global/documents/190883
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