|
Untitled 1
|
 |
ANNUAL CONFERENCE :: Abstract Library
Abstract Library
2003 Conference Abstract
| Type of Submission |
 |
Submission Type: |
Poster Presentation |
|
Subject Category: |
Structure and Function |
|
| Session Information |
|
Presentation Date: |
May 26, 2003 |
|
Abstract ID: |
F9 |
|
Session: |
Poster 1 |
|
Time: |
14:00 |
|
| Presenting Author |
|
| Other Authors |
|
P COURVILLE, INRS-Institut-Armand-Frappier
MFM CELLIER, INRS-Institut-Armand-Frappier
|
|
| Title |
|
Determination of E.coli MntH transmembrane topology |
|
| Abstract Text |
|
E.coli proton-dependent manganese transporter (MntH) facilitates uptake of Mn2+ and other divalent metals (Cd2+, Fe2+ and Co2+). E.coli MntH A (EcolA) protein belongs to one of three groups of bacterial homologs (MntH A, B, C) showing conservation of amino acid sequence and hydropathy profile with the eukaryotic Natural Resistance-Associated Macrophage Proteins (Nramp), which are chemiosmotic transporters facilitating proton-dependent uptake of divalent metals. MntH A proteins likely resemble Nramp ancestors and a consensus transmembrane (TM) topology was predicted including the N-terminus inside the cytoplasm followed by 11 or 12 TM segments. To test this prediction we studied EcolA protein and constructed protein fusions with ampicillin and chloramphenicol resistance reporters that indicate membrane sidedness (periplasm, ß-lactamase (Blam) and cytoplasm, chloramphenicol acetyltransferase (Cat), respectively). In-frame random Blam fusion proteins obtained after nested deletions of MntH sequence were screened for resistance to >100 µg/mL ampicillin; nine fusions were visualized by western blot of membrane extracts and the fusion points determined by DNA sequencing; six of them confirmed predicted periplasmic loops after TM segments 1, 5, 7, 9 and 11; however, three fusions indicated the loop after TM 10 as periplasmic. To resolve this discrepancy, EcolA-Blam hybrid proteins were generated and tested for both functions; two hybrids were bi-functional demonstrating periplasmic exposure of EcolA polypeptide chain after TM 7 and 11. The cytoplasmic exposure of the loops after TM 8 and 10 was demonstrated using two directed fusions that showed resistance to >8 µg/mL chloramphenicol. Together, the results demonstrate that EcolA spans the membrane 11 times with the N-terminus cytoplasmic, a large excess of positive charges in the cytoplasmic loops including topogenic signals, and the C-terminus periplasmic. This topological model is likely conserved among Nramp family. |
|
|
|
|
