Molecular cloning and sequencing of the sppA gene and characterization of the encoded protease IV, a signal peptide peptidase, of Escherichia coli.

Ichihara, Shigeyuki; Suzuki, T; Suzuki, Masami; Mizushima, Shôji

doi:10.1016/s0021-9258(18)67669-0

Cited by 59 publications

(4 citation statements)

References 34 publications

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“…The gene encoding signal peptide peptidase was isolated and found to encode a protein of 618 amino acids with a predicted molecular weight of 67241. 271 This correlated very well with the size of the purified protease IV isolated from cell lysate. The purification of protease IV involved the isolation of the envelope and detergent extraction of the membranes, followed by DE52 ion exchange chromatography and Sephacryl S-300 gel filtration.…”

Section: Signal Peptide Peptidasesupporting

confidence: 52%

“…Next, the gene encoding protease IV was isolated by screening the Clark and Carbon collection for clones overexpressing signal peptide peptidase activity. The gene encoding signal peptide peptidase was isolated and found to encode a protein of 618 amino acids with a predicted molecular weight of 67241 …”

Section: Signal Peptide Peptidasementioning

confidence: 99%

“…This correlated very well with the size of the purified protease IV isolated from cell lysate. The purification of protease IV involved the isolation of the envelope and detergent extraction of the membranes, followed by DE52 ion exchange chromatography and Sephacryl S-300 gel filtration . Finally, cross-linking studies showed that protease IV exists as a tetramer.…”

Section: Signal Peptide Peptidasementioning

confidence: 99%

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Signal Peptidases

et al. 2002

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Section: Signal Peptide Peptidasesupporting

confidence: 52%

Section: Signal Peptide Peptidasementioning

confidence: 99%

Section: Signal Peptide Peptidasementioning

confidence: 99%

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Signal Peptidases

et al. 2002

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“…In 1984, the protease that degrades the lipoprotein signal peptide, named signal peptide peptidase A (SppA), was purified to homogeneity (4) and shown to correspond to the previously identified inner membrane protein, protease IV (5). SppA was subsequently cloned and sequenced, and the gene-encoded protein was shown to be a tetramer (6). Sequence analysis of Escherichia coli SppA reveals three hydrophobic regions that are candidate membrane-spanning domains.…”

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confidence: 99%

Escherichia coli Signal Peptide Peptidase A Is a Serine-Lysine Protease with a Lysine Recruited to the Nonconserved Amino-Terminal Domain in the S49 Protease Family

et al. 2008

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Escherichia coli signal peptide peptidase A (SppA) is a serine protease which cleaves signal peptides after they have been proteolytically removed from exported proteins by signal peptidase processing. We present here results of site-directed mutagenesis studies of all the conserved serines of SppA in the carboxyl-terminal domain showing that only Ser 409 is essential for enzymatic activity. Also, we show that the serine hydrolase inhibitor FP-biotin inhibits SppA and modifies the protein but does not label the S409A mutant with an alanine substituted for the essential serine. These results are consistent with Ser 409 being directly involved in the proteolytic mechanism. Remarkably, additional site-directed mutagenesis studies showed that none of the lysines or histidine residues in the carboxyl-terminal protease domain (residues 326-549) is critical for activity, suggesting this domain lacks the general base residue required for proteolysis. In contrast, we found that E. coli SppA has a conserved lysine (K209) in the N-terminal domain (residues 56-316) that is essential for activity and important for activation of S409 for reactivity toward the FP-biotin inhibitor and is conserved in those other bacterial SppA proteins that have an N-terminal domain. We also performed alkaline phosphatase fusion experiments that establish that SppA has only one transmembrane segment (residues 29-45) with the C-terminal domain (residues 46-618) protruding into the periplasmic space. These results support the idea that E. coli SppA is a Ser-Lys dyad protease, with the Lys recruited to the amino-terminal domain that is itself not present in most known SppA sequences.

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Translocation of proteins across the membrane of the endoplasmic reticulum: A place for Saccharomyces cerevisiae

Larriba

1993

Yeast

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Molecular cloning and sequencing of the sppA gene and characterization of the encoded protease IV, a signal peptide peptidase, of Escherichia coli.

Cited by 59 publications

References 34 publications

Signal Peptidases

Signal Peptidases

Escherichia coli Signal Peptide Peptidase A Is a Serine-Lysine Protease with a Lysine Recruited to the Nonconserved Amino-Terminal Domain in the S49 Protease Family

Translocation of proteins across the membrane of the endoplasmic reticulum: A place for Saccharomyces cerevisiae

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