Localization to the inner surface of the cytoplasmic membrane by immunoelectron microscopy of enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli.
The phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli constitutes a major pathway for sugar translocation. It is composed of integral membrane proteins (enzyme II components) that recognize specific extracellular sugars as well as phosphocarrier proteins, one of which is called enzyme I. While enzyme I plays a role in energizing the enzyme II for sugar transfer, its precise cellular distribution had not previously been defined. This study was designed to elucidate the cellular location of this protein by immunoelectron microscopy. Enzyme I antibody bound to E. coli cryosections was visualized with protein A-gold. The gold particles in sections of wild-type E. coli were found primarily associated with the surface of the inner membrane. A strain of E. coli harboring a plasmid encoding the gene for enzyme I was also tested for its distribution of enzyme I. Consistent with the biochemically established overproduction of enzyme I, this strain showed an approximately 80-fold higher density of gold particles per unit cell volume than the wild-type cells. The substantial overproduction of immunoreactive enzyme I was associated with a significant (approximately 20-fold) increase in the amount of that protein bound to the inner membrane. In addition, a substantial fraction of the total enzyme I accumulated within a 60-nm-wide zone in the vicinity of the inner membrane. A model to explain the zonal distribution of enzyme I under conditions of overexpression of the protein is presented.
Ghosh
B K
BK
Robert Wood Johnson Medical School, Piscataway, NJ 08854.
Owens
K
K
Pietri
R
R
Peterkofsky
A
A
D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000648:ultrastructure; D005838:Genotype; D015151:Immunoblotting; D008854:Microscopy, Electron; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000032:analysis*
D010731:Phosphoenolpyruvate Sugar Phosphotransferase System