Fig. 3. A temperature- and ATP-dependent binding step after the initial binding of Ub conjugates to the proteasome leads to a tighter, salt resistant binding.
(A) Binding at 4°C is salt-sensitive. After the initial binding reaction, resin bound 26S were washed with increasing concentration of KCl (0-300 mM) at 4°C. Binding after washing without KCl was taken as 100%.
(B) Scheme for the binding assay to distinguish initial and tighter binding of Ub conjugates to proteasomes
(C) 26S purified from muscle were pre-bound to Poly-Ub-E6AP at 4°C. This initial binding (black bars) is completely reversed by high salt washes (300 mM NaCl). After the initial binding reaction, proteasomes were incubated for 30 min at 37°C or 4°C and washed with high salt. Resin bound proteasome activity after the second incubation was assayed, as described before and is represented by grey bars.
(D) The transition to the tighter binding of 26S to poly-Ub-E6AP was measured over time and compared to the stimulation of proteasomes. After a 30 min incubation at 4°C, the unbound fraction of proteasomes was removed and the resin was incubated at 37°C. The salt wash-resistant proteasome activity was monitored at the indicated times (0 - 40 min). The time course leading to the maximal extent of tight binding was compared with that for the stimulation of gate-opening (as monitored by the enhanced peptidase activity) after conjugate binding under the same conditions.
(E) Binding of 26S was performed as described in (C). After the initial binding, bound 26S were incubated at 37°C for 30 minutes in the presence of ATP, ADP or ATPγS. Resin bound proteasome activity after the second. Incubation was assayed as after washing with 300 mM NaCl and is represented in grey bars.
(F) The S5a UIM domain inhibits only the initial, but not the tighter binding of poly-Ub conjugates to proteasomes. Proteasome binding to poly-Ub-E6AP was measured with and without the addition of 5 μM of the UIM domain of S5a during or after the initial binding. The UIM domain inhibits the initial binding but when added after the initial binding has already occurred no significant reduction in the transition to the tighter binding was observed. The initial, salt sensitive binding after 30 min at 4°C is shown in black bars, the tighter, salt resistant binding is shown in grey bars. All values are the means of at least three independent experiments ± SEM.