Labeling of the ATP synthase of Escherichia coli from the head-group region of the lipid bilayer.
The isolated and membrane-bound forms of the adenosinetriphosphatase of Escherichia coli (ECF1 and ECF1F0, respectively) have been reacted with two lysine-specific reagents, sodium hexadecyl 4-[3H]formylphenyl phosphate (HFPP) and sodium methyl 4-[3H]formylphenyl phosphate (MFPP), and with the photoreactive reagent 1,2-[3H]dipalmitoyl-sn-glycerol 3-[[[(4-azido-2-nitrophenyl)amino]ethyl]-phosphate] (arylazidoPE). HFPP and arylazidoPE are amphipathic molecules, inserting by their hexadecyl moieties (one and two chains, respectively) into the lipid bilayer, with the reactive groups intercalated among the phospholipid head groups. MFPP is the water-soluble analogue of HFPP. The labeling patterns of ECF1F0 obtained with HFPP and arylazidoPE were very similar; in both cases the a and b subunits of the F0 part were the most heavily labeled polypeptides of the complex. Models of subunit a, arranged in six transmembrane helices, place most of the lysines in the head-group region, available for reaction with HFPP. Subunits alpha and beta of the ECF1 part were very poorly labeled in comparison to the a and b subunits, together incorporating only 4% as much HFPP and 7.5% as much arylazidoPE as the two F0 subunits together on a protein mass basis. Trypsin cleavage studies localized any labeling of the alpha subunit by arylazidoPE to the N-terminal 15 residues of this polypeptide. When MFPP was used, the alpha and beta subunits were very much more reacted than the F0 subunits. This implies that most of the mass of the alpha and beta subunits in ECF1F0 is above the membrane and not in contact with the bilayer surface.(ABSTRACT TRUNCATED AT 250 WORDS)
D001386:Azides / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology*; D008051:Lipid Bilayers; D008239:Lysine; D008954:Models, Biological; D009943:Organophosphorus Compounds / Q000378:metabolism*; D010712:Phosphatidic Acids / Q000378:metabolism; D010713:Phosphatidylcholines; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D014316:Tritium
D001386:Azides; D008051:Lipid Bilayers; D009943:Organophosphorus Compounds; D010712:Phosphatidic Acids; D010713:Phosphatidylcholines; D014316:Tritium; C054839:1,2-dipalmitoyl-sn-glycerol 3-((((4-azido-2-nitrophenyl)amino)ethyl)phosphate); C054838:hexadecyl 4-formylphenyl phosphate; C054837:methyl 4-formylphenyl phosphate; D006180:Proton-Translocating ATPases; D008239:Lysine