Protease IV, a cytoplasmic membrane protein of Escherichia coli, has signal peptide peptidase activity.

ABSTRACT The Escherichia coli cell division protein FtsQ is a central component of the divisome. FtsQ is a bitopic membrane protein with a large C-terminal periplasmic domain. In this work we investigated the role of the transmembrane segment (TMS) that anchors FtsQ in the cytoplasmic membrane. A set of TMS mutants was made and analyzed for the ability to complement an ftsQ mutant. Study of the various steps involved in FtsQ biogenesis revealed that one mutant (L29/32R;V38P) failed to functionally insert into the membrane, whereas another mutant (L29/32R) was correctly assembled and interacted with FtsB and FtsL but failed to localize efficiently to the cell division site. Our results indicate that the FtsQ TMS plays a role in FtsQ localization to the division site.

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Biochemical Properties of a Putative Signal Peptide Peptidase from the Hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1

Journal of Bacteriology ◽

10.1128/jb.187.20.7072-7080.2005 ◽

2005 ◽

Vol 187 (20) ◽

pp. 7072-7080 ◽

Cited By ~ 10

Author(s):

Rie Matsumi ◽

Haruyuki Atomi ◽

Tadayuki Imanaka

Keyword(s):

Substrate Specificity ◽

Signal Peptide ◽

Biochemical Characterization ◽

Transmembrane Domain ◽

Biochemical Properties ◽

Peptidase Activity ◽

Amino Acid Residues ◽

Hyperthermophilic Archaeon ◽

Signal Peptide Peptidase ◽

Putative Signal Peptide

ABSTRACT We have performed the first biochemical characterization of a putative archaeal signal peptide peptidase (SppATk) from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. SppATk, comprised of 334 residues, was much smaller than its counterpart from Escherichia coli (618 residues) and harbored a single predicted transmembrane domain near its N terminus. A truncated mutant protein without the N-terminal 54 amino acid residues (ΔN54SppATk) was found to be stable against autoproteolysis and was examined further. ΔN54SppATk exhibited peptidase activity towards fluorogenic peptide substrates and was found to be highly thermostable. Moreover, the enzyme displayed a remarkable stability and preference for alkaline pH, with optimal activity detected at pH 10. ΔN54SppATk displayed a Km of 240 ± 18 μM and a V max of 27.8 ± 0.7 μmol min−1 mg−1 towards Ala-Ala-Phe-4-methyl-coumaryl-7-amide at 80°C and pH 10. The substrate specificity of the enzyme was examined in detail with a FRETS peptide library. By analyzing the cleavage products with liquid chromatography-mass spectrometry, ΔN54SppATk was found to efficiently cleave peptides with a relatively small side chain at the P-1 position and a hydrophobic or aromatic residue at the P-3 position. The positively charged Arg residue was preferred at the P-4 position, while substrates with negatively charged residues at the P-2, P-3, or P-4 position were not cleaved. When predicted signal sequences from the T. kodakaraensis genome sequence were examined, we found that the substrate specificity of ΔN54SppATk was in good agreement with its presumed role as a signal peptide peptidase in this archaeon.

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FtsL, an Essential Cytoplasmic Membrane Protein Involved in Cell Division in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.174.23.7717.1992 ◽

1992 ◽

Vol 174 (23) ◽

pp. 7717-7728 ◽

Cited By ~ 60

Author(s):

Luz-Maria Guzman ◽

James J. Barondess ◽

Jon Beckwith

Keyword(s):

Escherichia Coli ◽

Cell Division ◽

Membrane Protein ◽

Cytoplasmic Membrane ◽

Topological Analysis ◽

Growth Conditions ◽

Bacterial Cell Division ◽

Leucine Zippers ◽

Protein Dimerization ◽

Membrane Spanning

We have identified a gene involved in bacterial cell division, located immediately upstream of the ftsI gene in the min 2 region of the Escherichia coli chromosome. This gene, which we named ftsL , was detected through characterization of Tn phoA insertions in a plasmid containing this chromosomal region. Tn phoA topological analysis and fractionation of alkaline phosphatase fusion proteins indicated that the ftsL gene product is a 13.6-kDa cytoplasmic membrane protein with a cytoplasmic amino terminus, a single membrane-spanning segment, and a periplasmic carboxy terminus. The ftsL gene is essential for cell growth and division. A null mutation in ftsL resulted in inhibition of cell division, formation of long, nonseptate filaments, ultimate cessation of growth, and lysis. Under certain growth conditions, depletion of FtsL or expression of the largest ftsL-phoA fusion produced a variety of cell morphologies, including Y-shaped bacteria, indicating a possible general weakening of the cell wall. The FtsL protein is estimated to be present at about 20 to 40 copies per cell. The periplasmic domain of the protein displays a sequence with features characteristic of leucine zippers, which are involved in protein dimerization.

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