Functional replacement of the hemolysin A transport signal by a different primary sequence.
Secretion of the 107-kDa hemolysin A (HlyA) from Escherichia coli is mediated by the membrane proteins hemolysin B and hemolysin D. Hemolysin B is a member of the so-called ATP binding cassette transporter superfamily, which includes the multidrug resistance P-glycoprotein, the cystic fibrosis CFTR protein, and the major histocompatibility complex-associated transporter of antigenic peptides. Recognition of HlyA by the hemolysin B/D transporter is dependent on a signal sequence mapped to the C-terminal 50 or so amino acids of the HlyA molecule. We show that the C-terminal 70 amino acids of leukotoxin from Pasteurella hemolytica can substitute functionally for the HlyA signal sequence. This 70-amino acid sequence contains no primary sequence similarity to the HlyA signal sequence; however, structural motifs of helix-turn-helix followed by strand-loop-strand can be deduced for both sequences. We also demonstrate by site-directed mutagenesis that changes to these predicted motifs affect transport function. It thus appears that the transport signal of HlyA may be defined by a higher-order structure and that the hemolysin transporter may recognize a much wider diversity of primary sequences than previously anticipated. This finding may have implications for understanding the basis of substrate specificity of other ATP binding cassette transporters.
Zhang
F
F
Division of Molecular and Structural Biology, University of Toronto, ON, Canada.
Greig
D I
DI
Ling
V
V
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism / Q000633:toxicity; D001427:Bacterial Toxins / Q000235:genetics; D001483:Base Sequence; D002352:Carrier Proteins / Q000378:metabolism; D004912:Erythrocytes / Q000187:drug effects / Q000502:physiology; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D005098:Exotoxins / Q000235:genetics*; D005798:Genes, Bacterial; D006460:Hemolysin Proteins / Q000235:genetics*; D006461:Hemolysis; D066298:In Vitro Techniques; D016978:Mannheimia haemolytica / Q000235:genetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D012756:Sheep
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D005098:Exotoxins; D006460:Hemolysin Proteins; C036507:Hlya protein, E coli; D009838:Oligodeoxyribonucleotides; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C018579:leukotoxin; D000255:Adenosine Triphosphate