In vivo reconstitution of an active siderophore transport system by a binding protein derivative lacking a signal sequence.
Transport of iron (III) hydroxamates across the inner membrane of Escherichia coli depends on a binding protein-dependent transport system composed of the FhuB, C and D proteins. The FhuD protein, which is synthesized as a precursor and exported through the cytoplasmic membrane, represents the periplasmic binding protein of the system, accepting as substrates a number of hydroxamate siderophores and the antibiotic albomycin. A FhuD derivative, carrying an N-terminal His-tag sequence instead of its signal sequence and therefore not exported through the inner membrane, was purified from the cytoplasm. Functional activity, comparable to that of wild-type FhuD, was demonstrated for this His-tag-FhuD in vitro by protease protection experiments in the presence of different substrates, and in vivo by reconstitution of iron transport in a fhuD mutant strain. The experimental data demonstrate that the primary sequence of the portion corresponding to the mature FhuD contains all the information required for proper folding of the polypeptide chain into a functional solute-binding protein. Moreover, purification of modified periplasmic proteins from the cytosol may be a useful approach for recovery of many polypeptides which are normally exported across the inner membrane and can cause toxicity problems when overproduced.
Rohrback
M R
MR
Universität Tübingen, Germany.
Paul
S
S
Köster
W
W
D000595:Amino Acid Sequence; D001483:Base Sequence; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D005291:Ferrichrome / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D033902:Periplasmic Binding Proteins; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism*
D002352:Carrier Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D033902:Periplasmic Binding Proteins; D021382:Protein Sorting Signals; C069969:fhuD protein, E coli; D005291:Ferrichrome; D006639:Histidine