Phospholipids occupy the internal lumen of the c ring of the ATP synthase of Escherichia coli.
The occupancy of the central cavity of the membrane-embedded c ring of the ATP synthase of Escherichia coli was investigated with a photo-cross-linking approach. Single cysteine mutants were created at c subunit positions 4, 8, and 11, which are oriented to the inside of the ring. These cysteines were alkylated with reagents carrying a photoactivatable substituent and illuminated. Subunit c and derivatives were then isolated and subjected to mass spectrometric analyses. The most noticeable product, which was found exclusively in irradiated samples, had a mass increase of 719 Da, consistent with a cross-link product between the substituted c subunit and phosphatidylethanolamine. Digestion with phospholipase C converted this product into one with a mass diminished by 126 Da, indicating that the phosphoethanolamine moiety was cleaved off. Hence, the cross-link forms to the diacylglycerol moiety of phosphatidylethanolamine. Control experiments showed that the subunit c-phospholipid adducts were formed in the ATP synthase complex in its natural membrane environment and were not artifacts arising from monomeric c subunits. We conclude therefore that the inner lumen of the c ring is occupied with phospholipids. No evidence was found for an extension of subunit a into this space.
Oberfeld
Benjamin
B
Institut für Mikrobiologie, ETH Zürich, 8093 Zürich, Switzerland.
Brunner
Josef
J
Dimroth
Peter
P
D025181:ATP Synthetase Complexes / Q000737:chemistry / Q000378:metabolism*; D000478:Alkylation; D001483:Base Sequence; D001665:Binding Sites; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000378:metabolism; D003545:Cysteine / Q000235:genetics; D004075:Diglycerides / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010714:Phosphatidylethanolamines / Q000737:chemistry / Q000378:metabolism; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism*; D010777:Photochemistry; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D010738:Type C Phospholipases / Q000378:metabolism
D003432:Cross-Linking Reagents; D004075:Diglycerides; D010714:Phosphatidylethanolamines; D010743:Phospholipids; D021122:Protein Subunits; C483858:phosphatidylethanolamine; D025181:ATP Synthetase Complexes; D010738:Type C Phospholipases; D003545:Cysteine