ISOLATION AND CHARACTERIZATION OF CLPX, A NEW ATP-DEPENDENT SPECIFICITY COMPONENT OF THE CLP PROTEASE OF ESCHERICHIA-COLI

We have used C-14-labeled bacteriophage lambdaO-DNA replication protei n as a probe to identify and purify Escherichia coli proteases capable of its degradation. In this manner, five different proteases (termed Lop) have been identified capable of degrading lambdaO protein to acid -soluble fragments in an ATP-dependent fashion. One of these activitie s was purified to homogeneity and shown to be composed of two differen t polypeptides. The 23,000-Da component (LopP) was identified as the p reviously characterized ClpAP protein, known to complex with ClpA to f orm the ClpAP, an ATP-dependent protease, capable of degrading casein. The second 46,000-Da component was identified as ClpX (LopC), coded b y a gene located in the same operon, but promoter distal to that codin g for ClpP (Gottesman, S., Clark, W. P., de Crecy-Lagard, V., and Maur izi, M. R. (1993) J. Biol. Chem. 268, 22618-22626). This identificatio n was based on the determination of the sequence of the first 24 amino acid residues of the purified ClpX protein and its identity with that predicted by the DNA sequence. The ClpXP protease is substrate specif ic, since it degrades casein (known to be degraded by ClpAP), lambdaP, or DnaK proteins slowly or not at all. These results suggest that Clp X protein directs ClpP protease to specific substrates. It is estimate d that 50% of all lambdaO-specific protease activity present in crude E. coli extracts is due to the ClpXP protease. We propose that transie nt inhibition of lambdaO degradation observed in vivo during the later stages of lambda-DNA replication in vivo is responsible for the switc h from bidirectional to unidirectional replication. One round unidirec tional replication will lead to strand separation resulting in a switc h from early (theta) to late (sigma) mode of lambda-DNA replication.