Mutations that improve export of maltose-binding protein in SecB- cells of Escherichia coli.
It previously has been proposed that the Escherichia coli SecB protein promotes the export of the maltose-binding protein (MBP) from the cytoplasm by preventing the folding of the precursor MBP (preMBP) into a translocation-incompetent conformation. The export of wild-type MBP is only partially blocked in SecB- cells. In contrast, the export of MBP16-1, an MBP species with a defective signal peptide, is totally dependent on SecB; hence, SecB- cells that synthesize MBP16-1 are unable to utilize maltose as a sole carbon source. The selection of Mal+ revertants primarily yielded mutants with alterations in the MBP16-1 signal peptide that permitted SecB-independent MBP export to the periplasm to various extents. Although each of these alterations increased the overall hydrophobicity of the signal peptide, it was not possible to strictly equate changes in hydrophobicity with the degree of SecB-independent export. Somewhat unexpectedly, two mutants were obtained in which MBP export in SecB- cells was markedly superior to that of the wild-type MBP. Although wild-type MBP is not cotranslationally translocated in SecB- cells, the two mutant proteins designated MBP172 and MBP173 exhibited significant cotranslational export in the absence of SecB. Thus, the role of SecB was partially supplanted by a signal peptide that promoted more rapid movement of MBP through the export pathway. When preMBP included the MBP172 signal peptide as well as an alteration in the mature moiety that slows folding, the SecB requirement for maximal MBP export efficiency was almost totally eliminated. These results provide additional strong support for the proposed antifolding role of SecB in MBP export.
Collier
D N
DN
Department of Microbiology and Immunology, School of Medicine, University of North Carolina, Chapel Hill 27599-7290.
Bassford
P J
PJ
D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D029968:Escherichia coli Proteins; D008040:Genetic Linkage; D005990:Glycerol / Q000378:metabolism; D007700:Kinetics; D008320:Maltose / Q000378:metabolism; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D009154:Mutation; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; C461599:maltose transport system, E coli; D008320:Maltose; D005990:Glycerol