Substitution of tyrosine for either cysteine in beta-lactamase prevents release from the membrane during secretion.

Fitts, Renee; Reuveny, Ziva; Amsterdam, J van; Mulholland, Jon; Botstein, David

doi:10.1073/pnas.84.23.8540

Cited by 33 publications

(19 citation statements)

References 24 publications

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“…While this experiment elegantly provides additional evidence for the requirement of SecD for protein translocation, it does not prove that SecD specifically functions in the release or folding of proteins. An identical intermediate has been observed when the activity of SecA, SecD, SecE, SecF, or SecY is compromised (34,43), when the proton motive force is disrupted (16,17) and when the folding of 13-lactamase or heat-labile enterotoxin is inhibited by mutations (13,39); it is absent only in secB::TnS mutants (22) and after MBP-P-galactosidase fusion protein induction (34). This release intermediate may actually represent two distinct intermediates, a translocation intermediate whose carboxy terminus resides within the cytoplasm and an exported release or folding intermediate.…”

Section: Discussionmentioning

confidence: 97%

Genetic and molecular characterization of the Escherichia coli secD operon and its products

1994

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Section: Discussionmentioning

confidence: 97%

Genetic and molecular characterization of the Escherichia coli secD operon and its products

1994

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“…There are no cons at position -2. The residues at positions + 1 and to result in different SDS-PAGE migration patterns (9,11). Finally, the cellular location of ,B-lactamase was determined for the wild type and mutants 5-4, 5-14, and 7-4.…”

Section: Resultsmentioning

confidence: 99%

“…Alanine also predominates at although other residues are also tolerated. In position of the cleavage site has shifted for mi [5][6][7][8][9][10][11][12][13][14]. Thus, the exact spacing of the cleavage site amino terminus and hydrophobic core of the sil not essential for efficient cleavage.…”

Section: Resultsmentioning

confidence: 99%

Selection of functional signal peptide cleavage sites from a library of random sequences

Palzkill

Wong

et al. 1994

J Bacteriol

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The export of proteins to the periplasmic compartment of bacterial cells is mediated by an amino-terminal signal peptide. After transport, the signal peptide is cleaved by a processing enzyme, signal peptidase I. A comparison of the cleavage sites of many exported proteins has identified a conserved feature of small, uncharged amino acids at positions -1 and -3 relative to the cleavage site. To determine experimentally the sequences required for efficient signal peptide cleavage, we simultaneously randomized the amino acid residues from positions -4 to +2 of the TEM-1 I-lactamase enzyme to form a library of random sequences. Mutants that provide wild-type levels of ampicillin resistance were then selected from the random-sequence library. The sequences of 15 mutants indicated a bias towards small amino acids. The N-terminal amino acid sequence of the mature enzyme was determined for nine of the mutants to assign the new -1 and -3 residues. Alanine was present in the -1 position for all nine of these mutants, strongly supporting the importance of alanine at the -1 position. The amino acids at the -3 position were much less conserved but were consistent with the -3 rules derived from sequence comparisons. Compared with the wild type, two of the nine mutants have an altered cleavage position, suggesting that sequence is more important than position for processing of the signal peptide.Many secreted proteins in prokaryotes and eukaryotes are synthesized with an amino-terminal extension referred to as the signal peptide. The signal peptide directs protein translocation across membranes and is removed by a membranebound signal peptidase enzyme after transit through the membrane.The signal peptide consists of three regions: a 1-to 5-residue amino-terminal positively charged segment, a 10-to 15-residue central hydrophobic core, and a 3-to 7-residue hydrophilic carboxy-terminal section (28). The carboxy-terminal domain is the site of signal peptide cleavage by the signal peptidase. There are two Escherichia coli signal peptidases. Signal peptidase II processes lipoproteins (10), and signal peptidase I processes other secretory proteins (4). Sequence comparisons of many cleavage sites indicate that there is no strong sequence homology. However, the sites have a common pattern of small amino acids at the -1 (Ala, Gly, and Ser) and -3 (Ala, Gly, Ser, Val, and Ile) residue positions (20,25). Results from site-directed mutagenesis of the -1 and -3 positions support the -1 and -3 sequence requirements inferred from the sequence comparisons (7,24).In addition to the sequence requirements at -1 and -3, the transition from the central hydrophobic region to the cleavage region (-6 to -4) often contains a helix-breaking residue, such as Pro, Ser, or Gly. Studies that support the importance of a helix-breaking residue include mutagenesis experiments in which amino acid substitutions of the -4 proline of ,B-lactamase and the -6 proline of the M13 procoat were shown to result in inefficient signal peptide processing (11,24 (18,19). Th...

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“…Failure to achieve the native state would keep the protein membranebound. In support of this model there is evidence for a transient membrane-bound state for at least two periplasmic proteins (Minsky et al, 1986;Fitts et al, 1987;Thom and Randall, 1988). The alternative possibility is that association with the membrane is an artefactual consequence of the inability to attain the native state; the protein is exported to the periplasm and Membranes of C6 pDQ4 induced for B subunit synthesis and labelled in the presence of 2.5 mM dithiothreitol were subjected to equilibrium sucrose density gradient centrifugation.…”

Section: Resultsmentioning

confidence: 93%

Reduced B Subunit of Heat‐Labile Enterotoxin Associates with Membranes In vivo

Hardy

Hedges

1996

European Journal of Biochemistry

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The B subunit of heat-labile enterotoxin, a periplasmic protein of Escherichia coli has an internal disulfide bond that forms after the protein has been exported. The presence of 2.5 mM dithiothreitol in the medium prevents the formation of the disulfide bond and this causes the protein to rapidly bind to membranes, preferentially but not exclusively to the cytoplasmic membrane. The binding is irreversible in vivo but chaotropic agents disrupt the association between the non-native B subunit and the membranes in vitro. The fact that the reduced B subunit binds to both the cytoplasmic and outer membranes that enclose the periplasm suggests that it is exported normally to the periplasm and then, because it is unable to form its native structure, adsorbs to membranes in the vicinity. This is confirmed by the finding that when synthesised by spheroplasts, in which the outer membrane is disrupted, the majority of reduced B subunit, which is not now confined in the periplasm, is' exported to the medium and is not associated with membranes.

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Substitution of tyrosine for either cysteine in beta-lactamase prevents release from the membrane during secretion.

Cited by 33 publications

References 24 publications

Genetic and molecular characterization of the Escherichia coli secD operon and its products

Genetic and molecular characterization of the Escherichia coli secD operon and its products

Selection of functional signal peptide cleavage sites from a library of random sequences

Reduced B Subunit of Heat‐Labile Enterotoxin Associates with Membranes In vivo

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