Membrane topology analysis of the Escherichia coli aromatic amino acid efflux protein YddG.
YddG is an inner membrane protein (IMP) that exports aromatic amino acids in Escherichia coli. Topology models of YddG produced by sequence-based analysis in silico have predicted the presence of 9 or 10 potential transmembrane segments. To experimentally analyze the membrane topology of YddG, we used randomly created fusions to β-lactamase (BlaM) as a reporter. The selection of such fusions under 50 μg/ml of ampicillin had to fit with the periplasmic location of the BlaM domain. Five periplasmic loops of YddG predicted by the 10-transmembrane (TM) helices model were identified via the characterization of 12 unique in-frame fusions distributed along the yddG coding region. To confirm the 10-TM helices model further, cytoplasmic regions of YddG were identified with the help of ZsGreen fluorescent protein as a reporter. The presence of four cytoplasmic regions and the cytoplasmic localization of the C-terminus were revealed. Therefore, a 10-TM helices topology with cytoplasmic locations of the N- and C-termini is supported. The present data confirm the 'positive-inside rule' for IMPs and the early results of other workers regarding the cytoplasmic location of the C-terminus of YddG. The pole-specific localization of YddG-ZsGreen in E. coli cells was detected by fluorescence microscopy.
Ajinomoto-Genetika Research Institute, Lomonosov Moscow State University, Moscow, Russia.
Larisa G
LG
Airich
Irina S
IS
Tsyrenzhapova
Olga V
OV
Vorontsova
Alexey V
AV
Feofanov
Vera G
VG
Doroshenko
Sergey V
SV
Mashko
D000595:Amino Acid Sequence; D026921:Amino Acid Transport Systems, Neutral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D024322:Amino Acids, Aromatic / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D018272:Porins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism
D026921:Amino Acid Transport Systems, Neutral; D024322:Amino Acids, Aromatic; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D018272:Porins; D011993:Recombinant Fusion Proteins; C558730:YddG protein, E coli; D001618:beta-Lactamases