Hemolysin transport in Escherichia coli. Point mutants in HlyB compensate for a deletion in the predicted amphiphilic helix region of the HlyA signal.
The alpha-hemolysin transporter of Escherichia coli, a member of the ATP-binding cassette transporter super-family, is responsible for secretion of the 107-kDa protein toxin HlyA across both membranes of the Gram-negative envelope in a single step. Secretion of HlyA is dependent on a signal sequence, which occupies the C-terminal 50-60 amino acids of HlyA. Previously, it was shown that point mutants in the transmembrane domain of the transporter HlyB could partially correct the transport defect caused by a deletion of the C-terminal 29 amino acids of HlyA. These suppressor mutations demonstrated a direct interaction between HlyA and HlyB. They also displayed suppressor effects on a broad spectrum of HlyA signal mutants. In the present study, we selected HlyB alleles that complemented an internal deletion of 29 amino acids in HlyA containing a predicted amphiphilic helix region immediately upstream from the previous deletion. This set of HlyB mutants identifies further sites in HlyB that modulate substrate specificity but display allele-specific effects on a range of HlyA signal mutants. The inability to isolate mutations with effects restricted to either half of the signal sequence suggests that the signal is not recognized in a modular fashion by the transporter but rather functions as an integrated whole. We also report the isolation of the first substrate specificity mutation, which lies within the ATP-binding domain of HlyB. This could support a model in which the region of the ATP-binding cassette between the two Walker consensus motifs involved in ATP binding interacts with either the substrate or the transmembrane domains.
Sheps
J A
JA
Division of Molecular and Structural Biology, Ontario Cancer Institute, Toronto, Canada.
Cheung
I
I
Ling
V
V
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017354:Point Mutation; D017384:Sequence Deletion
D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins; C036507:Hlya protein, E coli; C050837:Hlyb protein, Bacteria