Topography of lactose permease from Escherichia coli.
The topography of lactose permease, in native membrane vesicles and after reconstitution of the purified protein into proteoliposomes, has been investigated by labeling the membrane-embedded portions of the protein using photoactivatable, hydrophobic reagents and by labeling the exposed portions of the protein with water-soluble, electrophilic reagents. Some sites of modification have been localized in fragments of the protein produced by chemical and enzymatic cleavage. These define a number of hydrophilic loops and membrane-spanning regions and give some substance to topographic models of the permease. The N-terminal third of the molecule was labeled by three photoactivatable reagents (3-(trifluoromethyl)-3-m-iodophenyldiazirine and the phospholipid analogues 2-(aceto-(4-benzoylphenylether]-1-palmitoylphosphatidylcholine) and 2-(4-azido-2-nitrophenylaminoacetyl)-1-palmitoylphosphatidylcholin e) as well as the water soluble, electrophilic reagents. The C-terminal part of the molecule is labeled by the diazirine and, to a lesser extent, by the phospholipid analogues. It apparently has more nucleophilic groups accessible to water-soluble reagents than the N-terminal domain, in which the density of apparently unreactive ionizable residues proved to be unexpectedly high. The apparent lack of reactivity of some of these residues may be explained either by their being buried in the protein moiety within the membrane domain, or by their close association with other ionizable residues on the surface of the protein.
D001389:Azirines / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D005033:Ethylmaleimide / Q000494:pharmacology; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins; D010446:Peptide Fragments / Q000302:isolation & purification; D010777:Photochemistry; D011487:Protein Conformation; D027981:Symporters
D001389:Azirines; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D010446:Peptide Fragments; D027981:Symporters; C033174:3-(trifluoromethyl)-3-(3-iodophenyl)diazirine; C021499:lactose permease; D005033:Ethylmaleimide