Site-specific antibodies against the PrlA (secY) protein of Escherichia coli inhibit protein export by interfering with plasma membrane binding of preproteins.
Genetic evidence indicates that the PrlA (SecY) protein of Escherichia coli functions as a membrane integrated signal sequence receptor in protein "export"--i.e., in protein translocation across (or integration into) the plasma membrane. We have raised antibodies in rabbits against two synthetic peptides representing the hydrophilic N- or C-terminal region of PrlA. Using these antibodies as probes in cell fractionation experiments, we confirm that PrlA is an integral membrane protein of the plasma membrane of E. coli. Fab fragments prepared from each of the two antisera specifically inhibit protein export by interfering with the binding of preproteins to the plasma membrane. Inhibition of preprotein binding and export by Fab fragments was shown in a cell-free translocation system: precursors for LamB (an integral membrane protein) and for a truncated form of MalE (a periplasmic protein) were first synthesized in a membrane-depleted E. coli-derived cell-free translocation system followed by posttranslational incubation with inverted vesicles derived from the plasma membrane of E. coli. Our data thus indicate that the N and C termini of PrlA are exposed to the cytoplasm. We discuss the possibility that the transmembrane segments of PrlA could be arranged in the lipid bilayer in a cylindrical fashion, thereby delimiting a protein conducting channel, with a signal sequence binding domain represented, at least in part, by the N and C termini of PrlA. Such a channel would also contain a "stop-transfer" sequence binding domain that in response to a stop-transfer sequence would open the cylindrical channel to the lipid bilayer and permit displacement of the polypeptide from the channel to the lipid bilayer, resulting in membrane integration.
Watanabe
M
M
Laboratory of Cell Biology, Howard Hughes Medical Institute, Rockefeller University, New York, NY 10021.
Blobel
G
G
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000276:immunology / Q000378:metabolism* / Q000502:physiology*; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D005798:Genes, Bacterial; D015151:Immunoblotting; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D007158:Immunologic Techniques; D008565:Membrane Proteins / Q000502:physiology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000502:physiology
D001426:Bacterial Proteins; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D008565:Membrane Proteins; D010446:Peptide Fragments; D011498:Protein Precursors; D021382:Protein Sorting Signals