Chaperone-mediated degradation of ssrA-tagged substrates by ClpP1P2 requires the hydrophobic patch on ClpP2.

<p>ClpX and ClpC1-dependent degradation of model substrates was assayed with a set of mature ClpP1P2 particles, created from mixed wild-type (wtP1, wtP2) and hydrophobic patch variants (hpP1, hpP2) of ClpP1 and ClpP2. A. Degradation of MDH-ssrA (2 μM) mediated by ClpX (1 μM hexamer) and wt, hp or mixed mature ClpP1P2 particles (0.5 μM double-ring particle), was followed by the disappearance of the MDH-ssrA band in SDS-PAGE. The band just below MDH-ssrA that is not degraded (*) was confirmed by MS/MS to be composed of MDH, most probably lacking the ssrA tag. B. Degradation of GFP-ssrA (2 μM) mediated by ClpC1 (1 μM hexamer) and by wt, hp and mixed mature ClpP1P2 particles (0.5 μM double-ring particle) was monitored by the loss of the intrinsic GFP fluorescence signal. The signal was globally normalized. Additionally, time points were taken at the beginning and the end of the reaction and degradation of GFP-ssrA was confirmed by SDS-PAGE.