Complementation of transport-deficient mutants of Escherichia coli alpha-hemolysin by second-site mutations in the transporter hemolysin B.
Hemolysin B (HlyB) is a membrane-bound transport protein composed of an amino-terminal multiple membrane-spanning portion followed by a conserved ATP binding sequence. Together with the inner membrane protein HlyD and the outer membrane protein TolC, HlyB is responsible for transport of the 107-kDa toxin HlyA from the cytoplasm, across both membranes of the cell envelope of Escherichia coli, directly to the medium. We have used a mutational approach to investigate a postulated interaction between HlyA and HlyB. We have isolated transport-deficient mutants of HlyA altered in the C-terminal signal sequence and used one of these, a deletion of 29 amino acids, to select compensatory mutants in the transporter protein HlyB. Fifteen mutants located at six different sites, all mapping within the amino-terminal multiple membrane-spanning domain of HlyB, were identified. All of the mutations are clustered into three groups located close to the predicted inner face of the cytoplasmic membrane. We propose that these locations are close to sites on HlyB that interact with the C-terminal signal sequence of HlyA. This interaction is likely to involve either binding of HlyA to HlyB or activation of the transport mechanism. The compensatory mutants also display different patterns of specificity in terms of their ability to transport different HlyA mutants. The fact that point mutations are able to compensate for drastic changes in the signal sequence of HlyA suggests that substrate specificity of transporters such as HlyB may shift dramatically during evolutionary history. This could account for the diversity of substrates observed for the ABC transporter superfamily in nature.
Zhang
F
F
Division of Molecular and Structural Biology, University of Toronto, Ontario, Canada.
Sheps
J A
JA
Ling
V
V
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001692:Biological Transport / Q000235:genetics; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003062:Codon / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005816:Genetic Complementation Test; D006460:Hemolysin Proteins / Q000235:genetics / Q000378:metabolism*; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000633:toxicity; D007700:Kinetics; D008769:Methylnitronitrosoguanidine / Q000633:toxicity; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009153:Mutagens / Q000633:toxicity; D009838:Oligodeoxyribonucleotides; D017433:Protein Structure, Secondary; D015183:Restriction Mapping; D013489:Suppression, Genetic
D001426:Bacterial Proteins; D002352:Carrier Proteins; D003062:Codon; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins; C036507:Hlya protein, E coli; C050837:Hlyb protein, Bacteria; D006898:Hydroxylamines; D009153:Mutagens; D009838:Oligodeoxyribonucleotides; D008769:Methylnitronitrosoguanidine; D019811:Hydroxylamine