Determination of the cleavage site involved in C-terminal processing of penicillin-binding protein 3 of Escherichia coli

Nagasawa, Hiromich; Sakagami, Youji; Suzuki, A; Suzuki, Hirotoshi; Hara, Hiroshi; Hirota, Yukinori

doi:10.1128/jb.171.11.5890-5893.1989

Cited by 61 publications

(52 citation statements)

References 16 publications

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“…Fig. 4 (20). Since purified Tsp is a C-terminal-specific protease that can cleave after valine, it seems extremely likely that it is directly responsible for the C-terminal cleavage of penicillinbinding protein 3 that occurs in the cell.…”

Section: Discussionmentioning

confidence: 99%

Tsp: a tail-specific protease that selectively degrades proteins with nonpolar C termini.

Silber

Keiler

Sauer

1992

Proc. Natl. Acad. Sci. U.S.A.

186

192

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An Escherichia coli protease designated Tsp (tail-specific protease) has been purified, and its gene has been cloned and sequenced. Tsp specifically degrades a variant of the N-terminal domain of A repressor in which the five C-terminal residues, which are polar in wild type, have been replaced by nonpolar residues. This substrate specificity in vitro parallels the previously reported selective degradation in vivo of N-terminal-domain variants with nonpolar C-terminal residues. The gene sequence and N-terminal protein sequence of Tsp predict a protein of 660 amino acids. The deduced protein sequence of Tsp shows no significant homology to known protease sequences but does show sequence similarity to the human and bovine interphotoreceptor retinoid-binding proteins, which bind hydrophobic ligands.Within cells, different proteins are degraded with half-lives ranging from minutes to days (1). This heterogeneity implies that intracellular degradation is selective, but the mechanisms of this selectivity are incompletely understood. The C-terminal sequences of some proteins can have dramatic effects on their rates of degradation in Escherichia coli (2, 3). For example, the wild-type N-terminal domain of A repressor (residues 1-102) has the polar C-terminal amino acid sequence RSEYE102 and has a half-life in vivo of >10 hr. The rapidly degraded "#105" variant of this protein has the hydrophobic C-terminal sequence WVAAA102 and has a half-life of 15 min (3). To identify possible cellular component(s) responsible for the degradation of proteins with destabilizing C termini, we have purified an E. coli activity that specifically degrades the #105 variant but not the wild-type N-terminal domain. This activity resides in a single, purified polypeptide (Tsp, for tail-specific protease). We have cloned and sequenced the tsp gene.* Based on sequence comparisons, Tsp shows similarities to the human and bovine interphotoreceptor retinoid-binding proteins (IRBPs) but does not resemble any proteases in the protein sequence data base. MATERIALS AND METHODSAssays for Tsp Activity. Tsp activity in crude lysates and column fractions was assayed by measuring the degradation of 35S-labeled substrates (either the #105 variant or the wild-type N-terminal domain of A repressor) to products soluble in 10% (wt/vol) trichloroacetic acid (3). Reaction mixtures contained _9000 cpm of 35S-labeled substrate, a sample of the E. coli fraction in lysis buffer or column buffer, and 0.02% Nonidet P-40 in a volume of 50 p1. Reaction mixtures were incubated at 370C for 1 or 2 hr and were processed as described (3).In more purified fractions, degradation was assayed by the appearance of substrate digestion products following SDS/ PAGE. Wild-type or #105 protein (2 ,ug), which was purified by the method of Lim and Sauer (4), was mixed with a sample in a 10-1LI reaction volume. Reaction mixtures were incubated at 370C for 3-6 hr and were stopped by boiling for 3 min in Laemmli sample buffer (5). Samples were electrophoresed in SDS/16.5% polyacrylam...

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Section: Discussionmentioning

confidence: 99%

Tsp: a tail-specific protease that selectively degrades proteins with nonpolar C termini.

Silber

Keiler

Sauer

1992

Proc. Natl. Acad. Sci. U.S.A.

186

192

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“…One is a polyclonal antibody raised against the entire FtsI molecule. The other is a polyclonal antibody raised against a synthetic peptide corresponding to the last 14 amino acids of the mature FtsI polypeptide (residues 564-577) (Nagasawa et al, 1989). Both antibodies were affinity purified (Pringle et al, 1991) against the periplasmic domain of FtsI (Fraipont et al, 1994).…”

Section: Antibodies Against Ftsimentioning

confidence: 99%

“…FtsI is a cytoplasmic membrane protein with a simple structure: a small amino-terminal cytoplasmic domain (23 amino acids), a single membrane-spanning segment (17 amino acids) and a large periplasmic domain (536 amino acids) (Bowler and Spratt, 1989;Nagasawa et al, 1989) The periplasmic domain has a transpeptidase activity that is needed for synthesis of the peptidoglycan cell wall. There is one report that the periplasmic domain of FtsI also has transglycosylase activity (Ishino and Matsuhashi, 1981), but this is controversial (Ghuysen, 1994).…”

Section: Introductionmentioning

confidence: 99%

Localization of the Escherichia coli cell division protein FtsI (PBP3) to the division site and cell pole

Weiss

Pogliano

Carson

et al. 1997

Molecular Microbiology

107

100

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SummaryFtsI, also known as penicillin-binding protein 3, is a transpeptidase required for the synthesis of peptidoglycan in the division septum of the bacterium, Escherichia coli. FtsI has been estimated to be present at about 100 molecules per cell, well below the detection limit of immunoelectron microscopy. Here, we confirm the low abundance of FtsI and use immunofluorescence microscopy, a highly sensitive technique, to show that FtsI is localized to the division site during the later stages of cell growth. FtsI was also sometimes observed at the cell pole; polar localization was not anticipated and its significance is not known. We conclude (i) that immunofluorescence microscopy can be used to localize proteins whose abundance is as low as approximately 100 molecules per cell; and (ii) that spatial and temporal regulation of FtsI activity in septum formation is achieved, at least in part, by timed localization of the protein to the division site.

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“…Prc has been shown in vivo to be a protease that cleaves 11 residues from the C terminus of FtsI (penicillin-binding protein 3) (39). FtsI is an essential enzyme that is required for septum formation in the dividing cell (52).…”

Section: ϫ3mentioning

confidence: 99%

Multicopy suppressors of prc mutant Escherichia coli include two HtrA (DegP) protease homologs (HhoAB), DksA, and a truncated R1pA

Bass¹,

Gu²,

Christen³

1996

J Bacteriol

142

120

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We have isolated three multicopy suppressors of the conditional lethal phenotype of a prc (tsp) null strain of Escherichia coli. One of these suppressors included two novel putative protease genes in tandem that map to 3400 kb or 72.5 centisomes on the chromosome. We propose the names hhoA and hhoB, for htrA homolog, to denote that these genes encode proteins that are 58 and 35% identical, respectively, to the HtrA (DegP) serine protease and 36% identical to each other. The HhoA and HhoB proteins are predicted to be 455 and 355 amino acids, respectively, in length. The mature HhoA protein is periplasmic in location, and amino-terminal sequencing shows that it arises following cleavage of a 27-amino-acid signal peptide. Searches of the protein and DNA databases reveal a rapidly growing family of homologous genes in a variety of other bacteria, including several which are required for virulence in their host. Deletion of the hhoAB genes shows that they are not required for viability at high temperatures like the homologous htrA but grow more slowly than wild-type strains. A second multicopy prc suppressor is the dksA (dnaK suppressor) gene, which is also a multicopy suppressor of defects in the heat shock genes dnaK, dnaJ, and grpE. The dksA gene was independently isolated as a multicopy suppressor of a mukB mutation, which is required for chromosomal partitioning. A third dosage-dependent prc suppressor includes a truncated rare lipoprotein A (rlpA) gene.

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Determination of the cleavage site involved in C-terminal processing of penicillin-binding protein 3 of Escherichia coli

Cited by 61 publications

References 16 publications

Tsp: a tail-specific protease that selectively degrades proteins with nonpolar C termini.

Tsp: a tail-specific protease that selectively degrades proteins with nonpolar C termini.

Localization of the Escherichia coli cell division protein FtsI (PBP3) to the division site and cell pole

Multicopy suppressors of prc mutant Escherichia coli include two HtrA (DegP) protease homologs (HhoAB), DksA, and a truncated R1pA

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