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J Biol Chem, Vol. 274, Issue 20, 13999-14005, May 14, 1999

Recognition, Targeting, and Hydrolysis of the  O Replication Protein by the ClpP/ClpX Protease

Malgorzata Gonciarz-Swiatek, Alicja Wawrzynow, Soo-Jong Um^§, Brian A. Learn^§, Roger McMacken^§, William L. Kelley^¶, Costa Georgopoulos^¶, Olaf Sliekers, and Maciej Zylicz

From the  Department of Molecular and Cellular Biology, Faculty of Biotechnology, University of Gdansk, 80-822 Gdansk, Kladki 24, Poland, the ^§ Department of Biochemistry, School of Hygiene and Public Health, Johns Hopkins University, Baltimore, Maryland 21205, and the ^¶ Department de Biochimie Medicale, Centre Medical Universitaire, 1, rue Michel-Servet, 1211 Geneva 4, Switzerland

It has previously been established that sequences at the C termini of polypeptide substrates are critical for efficient hydrolysis by the ClpP/ClpX ATP-dependent protease. We report for the bacteriophage  O replication protein, however, that N-terminal sequences play the most critical role in facilitating proteolysis by ClpP/ClpX. The N-terminal portion of  O is degraded at a rate comparable with that of wild type O protein, whereas the C-terminal domain of O is hydrolyzed at least 10-fold more slowly. Consistent with these results, deletion of the first 18 amino acids of  O blocks degradation of the N-terminal domain, whereas proteolysis of the O C-terminal domain is only slightly diminished as a result of deletion of the C-terminal 15 amino acids. We demonstrate that ClpX retains its capacity to bind to the N-terminal domain following removal of the first 18 amino acids of O. However, ClpX cannot efficiently promote the ATP-dependent binding of this truncated O polypeptide to ClpP, the catalytic subunit of the ClpP/ClpX protease. Based on our results with  O protein, we suggest that two distinct structural elements may be required in substrate polypeptides to enable efficient hydrolysis by the ClpP/ClpX protease: (i) a ClpX-binding site, which may be located remotely from substrate termini, and (ii) a proper N- or C-terminal sequence, whose exposure on the substrate surface may be induced by the binding of ClpX.

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