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J A Sheps

Publications and source records attributed to J A Sheps.

4 recordsLinked to original sources
PubMed1995

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.

Amino Acid Sequence↗
PubMed1993

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.

Amino Acid Sequence↗