Figure 1.
Homotypic K11-Linked PolyUb Chains Do Not Bind Strongly to the 26S Proteasome
(A) Schematic shows truncated Ube2S, with the lysine rich C terminus removed, leaving a terminal lysine at position 197 (Ube2SΔ).
(B and C) Homotypic K11-polyUb conjugates are formed on Ube2SΔ. Resin-bound Ube2SΔ was incubated with E1, Ub, AMSH, and ATP for 4 hr at 37°C. The resins were washed and either analyzed by SDS-PAGE and Coomassie staining (B) or the Ub linkages measured by AQUA MS (C).
(D) K11-polyUb Ube2SΔ does not bind significantly to purified proteasomes. Polyubiquitinated E6AP and Ube2SΔ, or non-modified control resins, were incubated with purified 26S particles and the bound proteasomes were measured by LLVY-AMC cleavage.
(E) Homotypic K11-polyUb chains do not bind to the proteasome. Polyubiquitinated E6AP and Ube2SΔ, or non-modified control resins, were incubated with purified 26S particles and the bound proteasomes were measured by immunoblot for the 20S α subunits.
(F and G) K11-Ub4 does not compete with polyUb-E6AP to bind to the proteasome. Binding of 26S to PolyUb-E6AP in the presence of increasing concentrations of K48- or K11-Ub4 was measured. Binding of 26S proteasomes without the addition of Ub tetramers was taken as 100%.
Values are the means ± SEM of three (D) or four replicates (G).
E6, E6AP; 2S, Ube2SΔ; Ubn, polyUb chain of undefined length on the protein substrate.