Anti-peptide antibodies and proteases as structural probes for the lactose/H+ transporter of Escherichia coli: a loop around amino acid residue 130 faces the cytoplasmic side of the membrane.
From the amino acid sequence of the Escherichia coli lactose/H+ transporter, 7 hydrophilic segments were selected, 8-13 amino acids in length, and chemically synthesized, and anti-peptide antibodies were raised in rabbits. Apart from the antiserum to the synthetic COOH terminus (P408-417), which reacted strongly with the lactose/H+ transporter and has previously been used to localize the COOH terminus on the cytoplasmic face of the membrane, only those antibodies directed against the peptide corresponding to amino acid residues 125-135 (P125-135) exhibited a marked reaction with the transporter, while antibodies to the five other peptides reacted very weakly or not at all, suggesting that most of the hydrophilic segments are conformationally restricted or buried in the interior of the protein. Thermolysin treatment destroys the epitope on the transporter which is recognized by anti-P125-135 antibodies. Comparison of the kinetics and the extent of proteolysis of the transporter in right-side-out or inside-out cytoplasmic membrane vesicles or in reconstituted proteoliposomes suggests that the hydrophilic sequence from amino acid 125 to amino acid 135 is accessible to thermolysin only from one side, corresponding to the cytoplasmic face of the membrane. Furthermore, the experiments demonstrate that the transporter is inserted bimodally in a nonpreferential fashion into the proteoliposomes, confirming earlier results using antibodies to the synthetic COOH terminus of the transporter in conjunction with carboxypeptidase A treatment.
D000595:Amino Acid Sequence; D000906:Antibodies; D000936:Antigen-Antibody Complex; D002462:Cell Membrane / Q000201:enzymology; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins / Q000276:immunology; D009004:Monosaccharide Transport Proteins; D010447:Peptide Hydrolases; D010455:Peptides / Q000138:chemical synthesis; D011487:Protein Conformation; D027981:Symporters
D000906:Antibodies; D000936:Antigen-Antibody Complex; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D010455:Peptides; D027981:Symporters; C021499:lactose permease; D010447:Peptide Hydrolases