Identification of the gene lon (capR) product as a 94-kilodalton polypeptide by cloning and deletion analysis

Schoemaker, J M; Markovitz, Alvin

doi:10.1128/jb.147.1.46-56.1981

Cited by 29 publications

(21 citation statements)

References 34 publications

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“…Media and reagents. The media used have been described previously (14,47). NF, a drug that mimics UV radiation treatment (14,22,57), was purchased from Sigma Chemical Co. and used at a concentration of 2 ,ug/ml.…”

Section: Methodsmentioning

confidence: 99%

“…The bacterial strains used are listed in Table 1. The procedures for isolation of covalently closed plasmid DNA and for transformation of bacteria with plasmids were as previously described (47). For these studies we obtained two sulA+-containing plasmids from U. Henning.…”

Section: Methodsmentioning

confidence: 99%

“…Protein labeling and analysis by PAGE-SDS. Plasmidcontaining minicells were isolated and labeled with [35S]methionine as described previously (13,47). Whole cells with or without plasmids were induced with nalidixic acid and labeled with [35S]methionine as follows.…”

Section: Methodsmentioning

confidence: 99%

“…The lon+ gene has been cloned (47,57), and the protein has been purified to homogeneity (9,56). It is a tetramer (8) with subunits of 94 kilodaltons (kdal) that has multiple 552 SCHOEMAKER, GAYDA, AND MARKOVITZ because the Lon (CapR) protein is an ATP-dependent protease (9, 10; Charette, Ph.D. thesis) and mutations in lon affect the stability of proteins in vivo, it has been hypothesized that SulA protein is additionally regulated proteolytically via the Lon protease.…”

mentioning

confidence: 99%

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Regulation of cell division in Escherichia coli: SOS induction and cellular location of the sulA protein, a key to lon-associated filamentation and death

Schoemaker¹,

Gayda²,

Markovitz³

1984

J Bacteriol

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136

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Mutations in sulA (sfiA) block the filamentation and death of capR (ion) mutants that occur after treatments that either damage DNA or inhibit DNA replication and thereby induce the SOS response. Previous suiA-iacZ gene fusion studies showed that sulA is transcriptionally regulated by the SOS response system (lexAlrecA). SulA protein has been hypothesized to be additionally regulated proteolytically through the capR (lon) protease, i.e., in lon mutants lacking a functional ATP-dependent protease there would be more SulA protein. A hypothesized function for SulA protein is an inhibitor of cell septation. To investigate aspects of this model, we attempted to construct Ion, Ion sulA, and Ion sulB strains containing multicopy plasmids specifying the suiA+ gene. Multicopy suIA+ plasmids could not be established in Ion strains because more SulA protein accumulates than in a Ion' strain. When the sulA gene was mutated by a mini-Mu transposon the plasmid could be established in the Ion strains. In contrast, suiA+ plasmids could be established in lon+, lon sulA, and Ion sulB strains. The suiA+ plasmids caused Ion sulA and Ion sulB cells to exist as filaments without SOS induction and to be sensitive to UV light and nitrofurantoin. Evidence implicated higher basal levels of SulA protein in these Ion plasmid suiA + strains as the cause of filamentation. We confirmed that the SulA protein is an 18-kilodalton polypeptide and demonstrated that it was induced by treatment with nalidixic acid. The SulA protein was rapidly degraded in a lon+ strain, but was comparatively more stable in vivo in a lon sulB mutant. Furthermore, the SulA protein was localized to the membrane by several techniques. enzymatic activities: an ATP hydrolysis-dependent protease activity (9, 10; M. F. Charette, Ph.D. thesis, University of Chicago, 1981), DNA stimulated ATPase activity (7a), and nucleic acid-binding activity (56; Charette, Ph.D. thesis). In addition, a defective CapR protein (capR9 allele) has also been purified. The CapR9 protein has retained the general nucleic acid affinity (9, 56), but has lost the protease activity (8,9). In Ion mutants, second site mutations in sul (suppressor of Ion) prevent the filamentation and UV sensitivity without affecting the mucoid phenotype of the cells (14,17,26,27). These mutants were isolated as UV-, nitro-NF-, or methyl methanesulfonate-resistant derivatives of lon strains. The sul mutations are located at two loci on the E. coli chromosome; sulA is near pyrD (22 min), and sulB is near leu (2 min). sfiA and sfiB (sfi for suppressor of filament induction) are identical to sulA and suiB, respectively, and were isolated as spontaneous thermoresistant revertants of tif-1 (recA441) Ion strains (15,23,24). The tif-l Ion strains filament and die at 41°C. To account for their data, George et al. (15) proposed that a division inhibitor (product of the sulA or sulB gene?) was induced by UV and that it might be more stable in Ion strains.By means of a Mu d(amp-lac) operon fusion that linked the structural gene for 3...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Regulation of cell division in Escherichia coli: SOS induction and cellular location of the sulA protein, a key to lon-associated filamentation and death

Schoemaker¹,

Gayda²,

Markovitz³

1984

J Bacteriol

Self Cite

136

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“…Approximately two ATPs are utilized for each peptide bond cleaved in proteins [65]. The active enzyme is a tetramer which contains four identical subunits each with an M r 87 000 [95,127].…”

Section: Proteases Of E Colimentioning

confidence: 99%

Peptidases and proteases of Escherichia coli and Salmonella typhimurium

Lazdunski

1989

FEMS Microbiology Letters

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Structure of the catalytic domain of the human mitochondrial Lon protease: Proposed relation of oligomer formation and activity

et al. 2010

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ATP-dependent proteases are crucial for cellular homeostasis. By degrading short-lived regulatory proteins, they play an important role in the control of many cellular pathways and, through the degradation of abnormally misfolded proteins, protect the cell from a buildup of aggregates. Disruption or disregulation of mammalian mitochondrial Lon protease leads to severe changes in the cell, linked with carcinogenesis, apoptosis, and necrosis. Here we present the structure of the proteolytic domain of human mitochondrial Lon at 2 Å resolution. The fold resembles those of the three previously determined Lon proteolytic domains from Escherichia coli, Methanococcus jannaschii, and Archaeoglobus fulgidus. There are six protomers in the asymmetric unit, four arranged as two dimers. The intersubunit interactions within the two dimers are similar to those between adjacent subunits of the hexameric ring of E. coli Lon, suggesting that the human Lon proteolytic domain also forms hexamers. The active site contains a 3 10 helix attached to the N-terminal end of a-helix 2, which leads to the insertion of Asp852 into the active site, as seen in M. jannaschii. Structural considerations make it likely that this conformation is proteolytically inactive. When comparing the intersubunit interactions of human with those of E. coli Lon taken with biochemical data leads us to propose a mechanism relating the formation of Lon oligomers with a conformational shift in the active site region coupled to a movement of a loop in the oligomer interface, converting the proteolytically inactive form seen here to the active one in the E. coli hexamer.

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Identification of the gene lon (capR) product as a 94-kilodalton polypeptide by cloning and deletion analysis

Cited by 29 publications

References 34 publications

Regulation of cell division in Escherichia coli: SOS induction and cellular location of the sulA protein, a key to lon-associated filamentation and death

Regulation of cell division in Escherichia coli: SOS induction and cellular location of the sulA protein, a key to lon-associated filamentation and death

Peptidases and proteases of Escherichia coli and Salmonella typhimurium

Structure of the catalytic domain of the human mitochondrial Lon protease: Proposed relation of oligomer formation and activity

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