The Copper Efflux Regulator CueR Is Subject to ATP-Dependent Proteolysis in Escherichia coli

The trace element copper serves as cofactor for many enzymes but is toxic at elevated concentrations. In bacteria, the intracellular copper level is maintained by copper efflux systems including the Cue system controlled by the transcription factor CueR. CueR, a member of the MerR family, forms homodimers, and binds monovalent copper ions with high affinity. It activates transcription of the copper tolerance genes copA and cueO via a conserved DNA-distortion mechanism. The mechanism how CueR-induced transcription is turned off is not fully understood. Here, we report that Escherichia coli CueR is prone to proteolysis by the AAA+ proteases Lon, ClpXP, and ClpAP. Using a set of CueR variants, we show that CueR degradation is not altered by mutations affecting copper binding, dimerization or DNA binding of CueR, but requires an accessible C terminus. Except for a twofold stabilization shortly after a copper pulse, proteolysis of CueR is largely copper-independent. Our results suggest that ATP-dependent proteolysis contributes to copper homeostasis in E. coli by turnover of CueR, probably to allow steady monitoring of changes of the intracellular copper level and shut-off of CueR-dependent transcription.


Experimental procedures
CueR activity assays -E. coli ΔcueR, Φ(copA-lacZ) cells (Outten et al., 2000) were transformed with plasmids encoding constitutively expressed CueR, CueR_C112S or the empty vector (pACYC184). Cultures were grown in plastic ware in copper-free M9 minimal medium treated with 50 g/liter Chelex 100 resin (Bio-Rad) to remove trace metals. Before usage trace metals (without copper component) were added to the medium, mixed and sterile-filtered. Cells were grown to an optical density (A 580 ) of 0.5 at 30 °C, defined copper concentrations (CuSO 4 ) were added to the cultures for 1 h and 1 ml culture was harvested for β-galactosidase activity assay.
The assay was performed as described previously (Miller, 1972). Preparation of protein extracts and immunodetection -Cell pellets were resuspended in TE buffer depending on their optical density (10 mM Tris/HCl, pH 8; 1 mM EDTA; 50 μl TE buffer per A 580 of 1.0) and mixed with protein sample buffer (final concentrations of 2 % SDS (w/v),

In vivo degradation experiments
50 mM Tris/HCl, pH 6.8). Samples were incubated for 5 min at 95 °C, centrifuged (1 min, 16,000 × g) and subjected to SDS-PAGE and Western transfer using standard protocols (Sambrook & Russell, 2001). Strep-tagged fusion proteins were detected using a Strep-tag-HRP conjugate (IBA GmbH). Protein signals were visualized using Luminata Forte Western HRP substrate (Millipore) and the Chemi Imager Ready (Alpha Innotec). Half-lives of proteins were calculated by pixel counting with AlphaEaseFC software (version 4.0.0, Alpha Innotec).
In vitro degradation experiments -15 µM of Strep_CueR were incubated for 2 min at 37 °C in the degradation buffer described in (Bissonnette et al., 2010). In vitro degradation was initialized by addition of 20 mM ATP. Degradation experiments without addition of ATP were performed as controls. Results were visualized by SDS-PAGE and Western transfer using standard protocols (Sambrook & Russell, 2001).

Figure S1
Figure S1. Effect of increasing CuSO 4 concentrations on CueR activity. E. coli ΔcueR, Φ(copA-lacZ) cells were transformed with plasmids encoding constitutively expressed CueR, CueR_C112S or the empty vector (pACYC184) and were grown to exponential growth phase in M9 minimal medium at 30 °C. Cells were stressed with increasing CuSO 4 concentrations for 1 h and β-galactosidase activity was measured in Miller Units (MU). Standard deviations were calculated from at least two independent experiments.    Plasmid-encoded CueR variants were expressed for 20 min in exponential growth phase (M9 minimal medium; 30 °C). Translation was blocked by addition of Cm. Samples were taken at indicated time points, subjected to SDS-PAGE, Western transfer, and immunodetection. Halflives (T 1/2 ) and standard deviations were calculated from at least three independent experiments. For comparison the Western blot for Strep_CueR was taken from Figure 1C.

Figure S5. Strep_CueR is stable in vitro.
Strep_CueR was purified and used for a control in vitro degradation experiment without the presence of the Lon protease. The effect of ATP addition (+ATP) or the approach without ATP (-ATP) was analyzed. Samples were taken at indicated time points, subjected to SDS-PAGE, Western transfer, and immunodetection. Data are representative of two independent experiments.