Association of thioredoxin with the inner membrane and adhesion sites in Escherichia coli.
The intracellular localization of thioredoxin in Escherichia coli was determined by immunoelectron microscopy and correlated to previous biochemical data which had suggested that thioredoxin resides at inner-outer membrane adhesion sites. Since a considerable amount of thioredoxin was lost during preparation of cells for electron microscopy, we immobilized the protein with the heterobifunctional photoactivatable cross-linker p-azidophenacylbromide before the cells were fixed with aldehyde and embedded in Lowicryl K4M. Thin sections were labeled with affinity-purified antithioredoxin antiserum and protein A-gold complexes. Densities of immunolabel in a designated membrane-associated area and in the rest of the cytoplasm were compared and the data were statistically evaluated. Wild-type strain W3110 and strain SK3981, an overproducer of thioredoxin, exhibited increased labeling at the inner membrane and its adjacent cytoplasmic area. In contrast, the more centrally located cytoplasm of both strains showed much lower label density. This label distribution did not change with cell growth or in the stationary phase. Immunolabel was often found at bridges between the inner and outer membranes; this result is consistent with a model which places at least a portion of the thioredoxin at membrane adhesion sites, corresponding to an osmotically sensitive cytoplasmic compartment bounded by a hybrid inner-outer membrane (C.A. Lunn and V. Pigiet, J. Biol. Chem. 257:11424-11430, 1982; C.A. Lunn and V. Pigiet, J. Biol. Chem. 261:832-838, 1986). Specific label was absent in the periplasmic space.
Bayer
M E
ME
Bayer
M H
MH
Lunn
C A
CA
Pigiet
V
V
D001386:Azides; D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents; D004926:Escherichia coli / Q000378:metabolism* / Q000648:ultrastructure; D007163:Immunosorbent Techniques; D008854:Microscopy, Electron; D010777:Photochemistry; D013879:Thioredoxins / Q000378:metabolism*
D001386:Azides; D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D013879:Thioredoxins; C011171:4-azidophenacyl bromide