Copper-induced proteolysis of the CopZ copper chaperone of Enterococcus hirae.
The cop operon is a key element of copper homeostasis in Enterococcus hirae. It encodes two copper ATPases, CopA and CopB, the CopY repressor, and the CopZ metallochaperone. It was previously shown that the transcription of the operon is induced by copper. The concomitant increase in the levels of Cop proteins, particularly the CopB copper export ATPase, allows uncompromised growth of E. hirae in up to 5 mm ambient copper. We here show by Western blotting that the steady-state level of CopZ was increased only up to 0.5 mm copper. At higher copper concentrations, the level of CopZ was decreased and became undetectable at 5 mm media copper. When CopZ was overexpressed from a plasmid, the cells exhibited increased sensitivity to copper and oxidative stress, suggesting that high CopZ expression could become toxic to cells. In wild-type cells, the level of mRNA transcripts from the cop operon remained high in up to 5 mm copper, suggesting that CopZ was proteolyzed. Cell extracts were found to contain a copper-activated proteolytic activity that degraded CopZ in vitro. In this assay, Cu-CopZ was more susceptible to degradation than apo-CopZ. The growth of E. hirae in copper increased the copper-inducible proteolytic activity in extracts. Zymographic studies showed the presence of a copper-dependent protease in crude cell lysates. Thus, copper-stimulated proteolysis plays an important role in the regulation of copper homeostasis in E. hirae.
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Department of Clinical Pharmacology, University of Berne, Murtenstrasse 35, 3010 Berne, Switzerland.
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D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D003300:Copper / Q000378:metabolism*; D017931:DNA Primers; D016983:Enterococcus / Q000378:metabolism*; D006868:Hydrolysis; D018832:Molecular Chaperones / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015534:Trans-Activators / Q000378:metabolism*
D001426:Bacterial Proteins; C092379:CopZ protein, Enterococcus hirae; D017931:DNA Primers; D018832:Molecular Chaperones; D015534:Trans-Activators; D003300:Copper