Figure 5

Degradation of N- and C-terminally ssrA tagged variants of the 5₂-knotted protein UCH-L1 by ClpXP. (A) ClpXP degradation competition experiment with equimolar amounts of UCH-L1-ssrA (closed squares) and PR65/A-ssrA (closed circles) (5 µM each). Control reaction with PR65/A-ssrA (5 µM) without competitor shown as open circles. (B) Degradation of UCH-L1 with a crosslinked N-terminal ssrA-tag (ssrA-x-UCH-L1, 10 µM, closed diamonds). All reactions contained 0.5 µM ClpXΔN₆, 1 µM ClpP₁₄ and 4 mM ATP with regeneration system and were monitored by SDS-PAGE and densitometry. (C) Degradation of UCH-L1-ssrA variants by ClpXP (0.5 µM ClpXΔN₆, 1 µM ClpP₁₄) monitored by SDS-PAGE and densitometry: UCH-L1-ssrA wild type (10 µM, solid squares); F165A (9 µM, open circles), F162A (10 µM, open diamonds), ssrA-x-UCH-L1 (10 µM, closed diamonds). The reactions contained 4 mM ATP with a regeneration system.