title: Escherichia coli exports previously folded and biotinated protein domains.
abstract: Biotination of proteins is a post-translational modification that requires a folded acceptor domain. We previously showed that an acceptor domain fused to the carboxyl terminus of several cytosolic proteins results in biotinated fusion proteins in vivo. We now show that proteins encoded by translational gene fusions of two periplasmic proteins, alkaline phosphatase and TEM beta-lactamase, to carboxyl-terminal biotin-accepting sequences are biotinated and exported by Escherichia coli. Expression of the alkaline phosphatase fusion protein in wild type strains resulted in inefficient biotination of the fusion product. This result was due to the rapid export of the acceptor protein before biotination could occur since a very large increase in biotinated fusion protein levels was observed in strains lacking the SecB chaperone protein. The beta-lactamase fusion protein was biotinated but was only stable in strains lacking the DegP periplasmic protease. Both biotinated fusion proteins accumulated in the culture medium in strains possessing defective outer membranes. These results indicate that the export machinery can accommodate both a post-translational modification and a protein domain previously folded into its mature conformation in vivo.
authors.affiliation: Department of Microbiology, University of Illinois, Urbana 61801.
authors.forename: K E
authors.initials: KE
authors.lastname: Reed
authors.forename: J E
authors.initials: JE
authors.lastname: Cronan
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics; D001426:Bacterial Proteins / Q000378:metabolism; D001710:Biotin / Q000378:metabolism*; D002458:Cell Fractionation; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism*; D010957:Plasmids; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D001618:beta-Lactamases / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; C057842:SecB protein, Bacteria; D001710:Biotin; D000469:Alkaline Phosphatase; D001618:beta-Lactamases