Analysis of mutational alterations in the hydrophilic segment of the maltose-binding protein signal peptide.
Oligonucleotide-directed mutagenesis was employed to investigate the role of the hydrophilic segment of the Escherichia coli maltose-binding protein (MBP) signal peptide in the protein export process. The three basic residues residing at the amino terminus of the signal peptide were systematically substituted with neutral or acidic residues, decreasing the net charge in a stepwise fashion from +3 to -3. It was found that a net positive charge was not absolutely required for MBP export to the periplasm. However, export was most rapid and efficient when the signal peptide retained at least a single basic residue and a net charge of +1. The nature of the adjacent hydrophobic core helped to determine the effect of charge changes in the hydrophilic segment on MBP export, which suggested that these two regions of the signal peptide do not have totally distinct functions. Although the stepwise decrease in net charge of the signal peptide also resulted in a progressive decrease in the level of MBP synthesis, the data do not readily support a model in which MBP synthesis and export are obligately coupled events. The export defect resulting from alterations in the hydrophilic segment was partially suppressed in strains harboring certain prl alleles but not in strains harboring prlA alleles that are highly efficient suppressors of signal sequence mutations that alter the hydrophobic core.
Puziss
J W
JW
Department of Microbiology and Immunology, School of Medicine, University of North Carolina, Chapel Hill 27599-7290.
Fikes
J D
JD
Bassford
P J
PJ
D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D007700:Kinetics; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D009838:Oligodeoxyribonucleotides; D021382:Protein Sorting Signals / Q000235:genetics*; D012995:Solubility; D013329:Structure-Activity Relationship
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D009838:Oligodeoxyribonucleotides; D021382:Protein Sorting Signals; C461599:maltose transport system, E coli