Figure 1: Proteasome holoenzymes are loaded to MSNPN through non-covalent interactions and their activities are retained in the complex formation.

(a) Illustration of proteasome interaction with MSNPN. Affinity-purified human proteasomes with His-tagged β4 subunits were analysed by SDS–polyacrylamide gel electrophoresis (PAGE)/Coomassie brilliant blue (CBB) staining. See also Supplementary Figs 2 and 3 and for MSNPN and proteasome characterizations, respectively. *Proteasome-bound USP14. (b) Proteasomes were incubated with MSNPN using the indicated mass ratios. Samples were then subjected to spin-down and the pellet portions were used for SDS–PAGE and immunoblotting (IB). Input, proteasome alone with an equal amount as other proteasome–MSNPN complexes. Ptsm, 26S proteasome. (c) As in b except that 0.5 M imidazole was added to the reaction and the supernatant (Sup) and pellet fractions were separated for IB analysis. Short exp and long exp, short and long exposure of the blot, respectively. (d) TEM images of MSNPN and proteasome-charged MSNPN (Ptsm-MSNPN). (e) suc-LLVY-AMC hydrolysis assay using MSNPN, proteasomes, and proteasome–MSNPN complexes. The insert is the Michaelis–Menten plot of concentration-dependent suc-LLVY-AMC cleavage in the presence of proteasomes (0.4 nM) or proteasome–MSNPN complexes (1:50, 0.4 nM) for 15 min (r²>0.97). Each data point of the graphs shown represents the mean of triplicate determinations. r.f.u., relative fluorescence units. (f) In vitro proteasomal degradation assay using polyubiquitinated Sic1^PY (Ub-Sic1) with T7 tags. Reactions with proteasomes or proteasome–MSNPN complexes for the indicated times were analysed by SDS–PAGE/IB using anti-T7 and anti-USP14 antibodies.