Topology of the transposon Tn10-encoded tetracycline resistance protein within the inner membrane of Escherichia coli.
The transposon Tn10-encoded tetracycline resistance protein TetA is an integral membrane protein responsible for the export of tetracycline from the cytoplasmic to the periplasmic side of the inner membrane of Gram-negative bacteria. From a plot of the average hydrophobicity along the sequence of this protein, a two-dimensional membrane topology with 12 transmembrane domains may be predicted. Using plasmid-bearing Escherichia coli maxicells we specifically radiolabeled the TetA protein. The amino terminus of this membrane protein was shown not to be processed, and its location on the inner side of the cytoplasmic membrane was demonstrated by a newly developed use of a chemical method. Spheroplasts and inside-out vesicles of the TetA protein synthesizing maxicells were subjected to limited digestion by proteases of different specificities. The TetA protein was not accessible to proteases from the periplasmic side. On the inner side of the cytoplasmic membrane, the carboxyl terminus and four sites accessible to endoproteases could be identified. The cleavage sites are proposed to be localized between amino acid residues 60-70, 110-130, 180-200, and at amino acid 327. These results allow the definition of a model for the two-dimensional topology of the TetA protein.
Eckert
B
B
Institut für Biologie III, Albert-Ludwigs-Universität Freiburg, West Germany.
Beck
C F
CF
D000595:Amino Acid Sequence; D002268:Carboxypeptidases / Q000494:pharmacology; D043422:Carboxypeptidases A; D004251:DNA Transposable Elements; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics*; D006868:Hydrolysis; D008666:Metalloendopeptidases; D008969:Molecular Sequence Data; D011487:Protein Conformation; D012097:Repressor Proteins / Q000032:analysis*; D013753:Tetracycline Resistance / Q000235:genetics*; D014157:Transcription Factors / Q000032:analysis*; D014357:Trypsin / Q000494:pharmacology
D004251:DNA Transposable Elements; D012097:Repressor Proteins; D014157:Transcription Factors; C034811:tetracycline resistance-encoding transposon repressor protein; D002268:Carboxypeptidases; D010450:Endopeptidases; D043422:Carboxypeptidases A; D014357:Trypsin; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase