The two-component, ATP-dependent Clp protease of Escherichia coli. Purification, cloning, and mutational analysis of the ATP-binding component

The ATP-binding component (Component II, hereafter referred to as ClpA) of a two-component, ATP-dependent protease from Escherichia coli has been purified to homogeneity. ClpA is a protein with subunit Mr 81,000. It has an intrinsic ATPase activity and activates degradation of protein substrates onl...

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Bibliographic Details
Published in:The Journal of biological chemistry 1988-10, Vol.263 (29), p.15226-15236
Main Authors: Katayama, Y, Gottesman, S, Pumphrey, J, Rudikoff, S, Clark, W P, Maurizi, M R
Format: Article
Language:English
Subjects:
Adenosine Triphosphate - metabolism
Amino Acid Sequence
Analytical, structural and metabolic biochemistry
ATP-Dependent Proteases
Base Sequence
Biological and medical sciences
Chromatography, Gel
Chromatography, High Pressure Liquid
Chromatography, Ion Exchange
Cloning, Molecular
Enzymes and enzyme inhibitors
Escherichia coli
Escherichia coli - enzymology
Escherichia coli - genetics
Escherichia coli Proteins
Fundamental and applied biological sciences. Psychology
Genes
Genes, Bacterial
Genotype
Heat-Shock Proteins
Hydrolases
Kinetics
Macromolecular Substances
Molecular Sequence Data
Mutation
Protease La
Protein Binding
protein Clp
proteinase
purification
Serine Endopeptidases - genetics
Serine Endopeptidases - isolation & purification
Serine Endopeptidases - metabolism
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Summary:The ATP-binding component (Component II, hereafter referred to as ClpA) of a two-component, ATP-dependent protease from Escherichia coli has been purified to homogeneity. ClpA is a protein with subunit Mr 81,000. It has an intrinsic ATPase activity and activates degradation of protein substrates only in the presence of a second component (Component I, hereafter referred to as ClpP), Mg2+, and ATP. The amount of ClpA varies by less than a factor of 2 in cells grown in different media and at temperatures from 30 to 42 degrees C. ClpA does not appear to be a heat-shock protein since its synthesis is not dependent on htpR. Antibodies against purified ClpA were used to identify lambda transducing phage bearing the clpA gene. The cloned gene contains a DNA sequence expected to code for the first 28 amino acids of ClpA, which were determined by protein sequencing of purified ClpA. The clpA gene in the phage was mutated by insertion of delta kan defective transposons and the mutations were transferred to E. coli by homologous recombination. The clpA gene was mapped to 19 min on the E. coli chromosome. Mutant cells with insertions early in the gene produce no ClpA protein detectable in Western blots, and extracts of such mutant cells have no detectable ClpA activity. clpA- mutants grow well under all conditions tested and are not defective in turnover of proteins during nitrogen starvation nor in the turnover of such highly unstable proteins as the lambda proteins O, N, and cII, or the E. coli proteins SulA, RcsA, and glutamate dehydrogenase. The degradation of abnormal canavanine-containing proteins is defective in clpA mutants especially in cells that also have a lon- mutation. Extracts of clpA- lon- cells have ATP-dependent casein degrading activity.
ISSN:0021-9258
1083-351X
DOI:10.1016/S0021-9258(18)68168-2