title: Mutational analysis supports a role for multiple structural features in the C-terminal secretion signal of Escherichia coli haemolysin.
abstract: We have carried out an extensive mutational analysis of the C-terminal signal which targets the export of the 1024-residue haemolysin protein (HlyA) of Escherichia coli across both bacterial membranes into the surrounding medium. Over 60 variants of the HlyA C-terminal 53-amino-acid sequence were created by oligonucleotide-directed mutagenesis and fused to the HlyA N-terminal 830 residues. Transport of the HlyA derivatives by the HlyB/HlyD system was compared with the wild-type level and the data indicate that the HlyA C-terminal export signal lies within the last 48 amino acids and comprises three functional domains: an amphipathic, charged helix between residues 1,977 and R,996; a 13-amino-acid uncharged region from residue T,997 to S,1009; and an 8-amino-acid hydroxylated tail at the extreme C-terminus. Analogous features were found in the C-terminal sequences of an extended family of haemolysins, leukotoxins and proteases which are secreted by HlyB/HlyD-type translocators. In particular, all nine proteins which are secreted into the extracellular medium possess potential extended amphipathic helices. These results suggest a possible role for multiple regions of the HlyA C-terminal export signal in which the first two domains span the membranes and the third domain remains in the cytoplasm.
authors.affiliation: Department of Pathology, Cambridge University, UK.
authors.forename: P
authors.initials: P
authors.lastname: Stanley
authors.forename: V
authors.initials: V
authors.lastname: Koronakis
authors.forename: C
authors.initials: C
authors.lastname: Hughes
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D014644:Genetic Variation; D006460:Hemolysin Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011487:Protein Conformation; D021382:Protein Sorting Signals / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins; C036507:Hlya protein, E coli; D009838:Oligodeoxyribonucleotides; D021382:Protein Sorting Signals