Maturation of Escherichia coli maltose-binding protein by signal peptidase I in vivo. Sequence requirements for efficient processing and demonstration of an alternate cleavage site

• Published in: The Journal of biological chemistry 1990-02, Vol.265 (6), p.3417-3423
• Main Authors: Fikes, J D, Barkocy-Gallagher, G A, Klapper, D G, Bassford, P J
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; ATP-Binding Cassette Transporters; Base Sequence; Carrier Proteins - genetics; Carrier Proteins - isolation & purification; Endopeptidases - metabolism; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli Proteins; Maltose - metabolism; maltose-binding protein; Maltose-Binding Proteins; Membrane Proteins - genetics; Molecular Sequence Data; Monosaccharide Transport Proteins; Mutation; Oligonucleotide Probes; Protein Precursors - genetics; Protein Precursors - metabolism; Protein Processing, Post-Translational; Serine Endopeptidases; signal peptidase I
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)39783-2

Comparative analyses of a number of secretory proteins processed by eukaryotic and prokaryotic signal peptidases have identified a strongly conserved feature regarding the residues positioned -3 and -1 relative to the cleavage site. These 2 residues of the signal peptide are thought to constitute a recognition site for the processing enzyme and are usually amino acids with small, neutral side chains. It was shown previously that the substitution of aspartic acid for alanine at -3 of the Escherichia coli maltose-binding protein (MBP) signal peptide blocked maturation by signal peptidase I but had no noticeable effect or MBP translocation across the cytoplasmic membrane of its biological activity. This identified an excellent system in which to undertake a detailed investigation of the structural requirements and limitations for the cleavage site. In vitro mutagenesis was used to generate 14 different amino acid substitutions at -3 and 13 different amino acid substitutions at -1 of the MBP signal peptide. The maturation of the mutant precursor species expressed in vivo was examined. Overall, the results obtained agreed fairly well with statistically derived models of signal peptidase I specificity, except that cysteine was found to permit efficient processing when present at either -3 and -1, and threonine at -1 resulted in inefficient processing. Interestingly, it was found that substitutions at -1 which blocked processing at the normal cleavage site redirected processing, with varying efficiencies, to an alternate site in the signal peptide represented by the Ala-X-Ala sequence at positions -5 to -3. The substitution of aspartic acid for alanine at -5 blocked processing at this alternate site but not the normal site. The amino acids occupying the -5 and -3 positions in many other prokaryotic signal peptides also have the potential for constituting alternate processing sites. This appears to represent another example of redundant information contained within the signal peptide.