Topology of the ExbB protein in the cytoplasmic membrane of Escherichia coli.
The ExbB protein together with the ExbD and TonB proteins is involved in energy-coupled transport across the outer membrane of Escherichia coli. To understand this unusual process it is required to determine the subcellular location of ExbB and its transmembrane arrangement. Using ExbB-beta-lactamase fusion proteins as reporters for a periplasmic versus a cytoplasmic location of the fusion sites, and accessibility of ExbB in spheroplasts and cell lysates to aminopeptidase K, trypsin, and proteinase K, we arrived at a model of ExbB topology in the cytoplasmic membrane. Starting with the N terminus in the periplasm ExbB contains three transmembrane segments (residues 16-39, 128-155, 162-194) a small periplasmic loop and two large portions in the cytoplasm. Two of the 18 fusion proteins studied, ExbB34-beta-lactamase and ExbB41-beta-lactamase, conferred a high ampicillin resistance. Protease experiments revealed a high respectively low percentage of the molecules in a reverse transmembrane orientation. Both proteins were lacking positive charges at the inner side of the cytoplasmic membrane which determine the orientation of transmembrane segments.
Kampfenkel
K
K
University of Tübingen, Germany.
Braun
V
V
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001483:Base Sequence; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D003593:Cytoplasm; D004277:DNA, Single-Stranded; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins; D006868:Hydrolysis; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D001618:beta-Lactamases / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; C052874:ExbB protein, E coli; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; C060792:tolQ protein, E coli; C060794:tolR protein, E coli; D001618:beta-Lactamases