Export of unprocessed precursor maltose-binding protein to the periplasm of Escherichia coli cells

Abstract

The Escherichia coli maltose-binding protein (MBP) R2 signal peptide is a truncated version of the wild-type structure that still facilitates very efficient export of MBP to the periplasm. Among single amino acid substitutions in the R2 signal peptide resulting in an export-defective precursor MBP (pMBP) were two that replaced residues in the consensus Ala-X-Ala sequence (residues -3 to -1) that immediately precedes the cleavage site. It was suggested that the functional hydrophobic core and signal peptidase recognition sequence of this signal peptide substantially overlap and that these two alterations affect both pMBP translocation and processing. In this study, the export of pMBP by the mutants, designated CC15 and CC17, with these two alterations was investigated further. The pMBP of mutant CC17 has an Arg substituted for Leu at the -2 position. It was found that CC17 cells exported only a very small amount of MBP, but that which was exported appeared to be correctly processed. This result was consistent with other studies that have concluded that virtually any amino acid can occupy the -2 position. For mutant CC15, which exhibits a fully Mal+ phenotype, an Asp is substituted for the Ala at the -3 position. CC15 cells were found to export large quantities of unprocessed, soluble pMBP to the periplasm, although such export was achieved in a relatively slow, posttranslational manner. This result was also consistent with other studies that suggested that charged residues are normally excluded from the -3 position of the cleavage site. Using in vitro oligonucleotide-directed mutagenesis, we constructed a new signal sequence mutant in which Asp was substituted for Arg at the -3 position of an otherwise wild-type MBP signal peptide. This alteration had no apparent effect on pMBP translocation across the cytoplasmic membrane, but processing by signal peptidase was inhibited. This pMBP species with its full-length hydrophobic core remained anchored to the membrane, where it could still participate in maltose uptake. The implications of these results for models of protein export are discussed.