Labeling of the ATP synthase of Escherichia coli from the head-group region of the lipid bilayer

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Abstract
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 ampiphathic 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 the 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. One subunit of ECF1, the .delta. subunit, was heavily labeled by HFPP and MFPP in free ECF1 but was shielded from reaction with both of these probes and with arylazidoPE in ECF1F0 preparations. The implication is that the and subunit, a link between ECF1 and F0, is shielded by protein-protein interactions, at least in the lipid head-group region of the bilayer.