Isolation and characterization of ClpX, a new ATP-dependent specificity component of the Clp protease of Escherichia coli.

We have used 14C-labeled bacteriophage lambda O-DNA replication protein 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 lambda O protein to acid-soluble fragments in an ATP-dependent fashion. One of these activities was purified to homogeneity and shown to be composed of two different polypeptides. The 23,000-Da component (LopP) was identified as the previously characterized ClpP protein, known to complex with ClpA to form the ClpAP, an ATP-dependent protease, capable of degrading casein. The second 46,000-Da component was identified as ClpX (LopC), coded by a gene located in the same operon, but promoter distal to that coding for ClpP (Gottesman, S., Clark, W. P., de Crecy-Lagard, V., and Maurizi, M. R. (1993) J. Biol. Chem. 268, 22618-22626). This identification 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 specific, since it degrades casein (known to be degraded by ClpAP), lambda P, or DnaK proteins slowly or not at all. These results suggest that ClpX protein directs ClpP protease to specific substrates. It is estimated that 50% of all lambda O-specific protease activity present in crude E. coli extracts is due to the ClpXP protease. We propose that transient inhibition of lambda O degradation observed in vivo during the later stages of lambda-DNA replication in vivo is responsible for the switch from bidirectional to unidirectional replication. One round unidirectional replication will lead to strand separation resulting in a switch from early (theta) to late (sigma) mode of lambda-DNA replication.