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. Author manuscript; available in PMC: 2016 Jul 2.
Published in final edited form as: Mol Cell. 2015 Jun 11;59(1):104–116. doi: 10.1016/j.molcel.2015.05.014

Figure 3. CpdR binding to the ClpX N-terminal domain forms a recruitment interface.

Figure 3

Postulated models (box) for NTDClpX as a simple anchoring site for CpdR (left) or a unique partner in forming a composite recruitment interface for binding PdeA (right). (A) Cartoons illustrate the use of a rapamycin(rap)-induced FRB/FKBP dimer to tether CpdR or SspB (residues 10–125 that binds ssrA motif) directly to ΔN-ClpX. GFP-ssrA-SS (requires SspB for delivery) degradation by FKBP-ΔN-ClpXP when mediated by SspB-FRB. PdeA levels in the presence of FRB-CpdR/FKBP-ΔN-ClpXP/rap (detected by Western using α-PdeA due to overlapping bands). (B) PdeA levels when directly tethered to ΔN-ClpX via the FRB/FKBP dimer system (detected by Coomassie staining). FRB-PdeA C-terminus Arg-Gly is mutated to Asp-Asp to create FRB-PdeADD. (C) Impact of NTDClpX on PdeA levels in the presence of FRB-CpdR/FKBP-ΔN-ClpXP/rap (detected by α-PdeA Western blot). (D) Effect of NTDClpX fragment concentration on the delivery of GFP-PdeA to FRB-CpdR/FKBP-ΔN-ClpXP (rapamycin added). Apparent Kactivation is shown, see Methods. (E) Effect of phosphorylation on the NTDClpX-dependent delivery of GFP-PdeA to FRB-CpdR/FKBP-ΔN-ClpXP (rapamycin added). FRB-CpdR phosphorylated using its cognate phosphorelay CckA/ChpT (Biondi et al., 2006; Chen et al., 2009). (D&E) are represented as mean ± SD, n = 3 experiments. See also Figure S3.