| Autor: |
Larisa G, Airich, Irina S, Tsyrenzhapova, Olga V, Vorontsova, Alexey V, Feofanov, Vera G, Doroshenko, Sergey V, Mashko |
| Rok vydání: |
2010 |
| Předmět: |
|
| Zdroj: |
Journal of molecular microbiology and biotechnology. 19(4) |
| ISSN: |
1660-2412 |
| Popis: |
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. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
|
