Site-directed mutagenesis of the hydrogenase signal peptide consensus box prevents export of a beta-lactamase fusion protein.
A secretion vector, pVN1, expressing the [NiFe] hydrogenase signal peptide of Desulfovibrio vulgaris Hildenborough fused to beta-lactamase from Escherichia coli was constructed in order to study the unusual characteristics of hydrogenase signal peptides, which share a strictly conserved sequence, the consensus box: R-R-X-F-X-K. Although the hydrogenase signal peptide-beta-lactamase fusion protein was processed much more slowly than the fusion of beta-lactamase with its own signal peptide, the system mimicked several features expected for hydrogenase biosynthesis in E. coli, including increased export under anaerobic conditions. Site-directed mutagenesis of R(-28), the first arginine residue of the consensus box, to a glutamate completely inhibited export and processing of the fusion protein. The same mutation of R(-33), located outside the consensus box, had almost no effect. The data indicate a specific role for the consensus box sequence in the export mechanism for hydrogenase.
Nivière
V
V
Department of Biological Sciences, University of Calgary, Alberta, Canada.
Wong
S L
SL
Voordouw
G
G
D000595:Amino Acid Sequence; D001483:Base Sequence; D001692:Biological Transport; D016384:Consensus Sequence; D004252:DNA Mutational Analysis; D003901:Desulfovibrio / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006864:Hydrogenase / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism
D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D006864:Hydrogenase; D001618:beta-Lactamases