Abstract
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.
Highlights
From the $Laboratory of Molecular Biology and the VLaboratoryof Genetics, Division of Cancer Biology and Diagnosis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892
The clpA gene was mapped to 19 min on the E. coli chromosome
Inhibitors and uncouplers of clpA- mutants grow well under all conditions tested oxidative phosphorylation added to cell cultures block degraand are not defective in turnover of proteins during dation of rapidly degraded proteins in growing cells [8]and nitrogen starvation nor in the turnovoefrsuch highly of selective proteins degraded during nutrientdeprivation (9, unstable proteinsas the X proteins 0,N, and cII, or the 10)
Summary
From the $Laboratory of Molecular Biology and the VLaboratoryof Genetics, Division of Cancer Biology and Diagnosis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892. Mutant cells with insertionsearly in the gene produce no ClpA ples of proteolytic inactivation of enzymes in other microorganisms upon nutritional shifts have been reported [6, 7]. Another feature of intracellular proteolysis in E. coli, as well as in other prokaryotes and in eukaryotes, is that the protein detectable in Western blots, and extracts of rate-limiting stepin protein degradation in vivo appears to be such mutant cells have no detectable ClpA activity. Inhibitors and uncouplers of clpA- mutants grow well under all conditions tested oxidative phosphorylation added to cell cultures block degraand are not defective in turnover of proteins during dation of rapidly degraded proteins in growing cells [8]and nitrogen starvation nor in the turnovoefrsuch highly of selective proteins degraded during nutrientdeprivation (9, unstable proteinsas the X proteins 0,N, and cII, or the 10)
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