The in vivo assembly and function of the N- and C-terminal halves of the Tn10-encoded TetA protein in Escherichia coli.
The tetA gene was cut into its N- and C-terminal halves at the central EcoRI site and the two halves were subcloned individually or together under a separate lac promoter/operator. The expression of the C-terminal half was detected with a C-terminal-specific antibody. The amount of the N-terminal half in the cytoplasmic membrane was not affected by the presence of the C-terminal half. In contrast, the amount of the C-terminal half in the membrane was increased in the presence of the N-terminal half, indicating that the N-terminal half helps the stable folding of the C-terminal half in the membrane. Each half individually showed no tetracycline transport activity, however, when both halves were expressed together, the resultant complex showed about 40% of the tetracycline transport activity of the wild-type per number of the C-terminals of TetA protein in the membrane.
Division of Microbial Chemistry, Faculty of Pharmaceutical Sciences, Chiba University, Japan.
A
A
Yamaguchi
Y
Y
Someya
T
T
Sawai
D000595:Amino Acid Sequence; D017920:Antiporters; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004251:DNA Transposable Elements / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D007763:Lac Operon / Q000235:genetics; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D013752:Tetracycline / Q000378:metabolism*
D017920:Antiporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D004251:DNA Transposable Elements; D011993:Recombinant Fusion Proteins; C079051:tetA protein, Bacteria; D013752:Tetracycline