FIGURE 3. An NMR cytosolic factor confers resistance to mammalian and yeast proteasomes from competitive inhibition.

(A)Top panel: Schematic showing the design of the cross over experiment set up to evaluate if species differences in proteasome resistance to inhibition reflect an intrinsic property of the proteasome or the presence of a protective cytosolic factor. See Crossover Assays in Methods below for details. Bottom panel: When Prot_Ms were resuspended in SN_NMR they showed elevated activity and inhibition resistance, whereas Prot_NMR exposed to SN_MS displayed both lower activity and greater sensitivity to inhibition. Bars A and D represents the activity of reconstituted MS and NMR cytosols (A = percentage set to 100% activity and D= ChT-L activity per mg lysate) in the absence of any inhibitor. B and E reveal the change in activity following treatment of the reconstituted cytosols with 20 μM MG132 and indicate that in comparison with mouse, the proteasome in NMR cytosol is resistant to inhibition. Bars C and F demonstrate that when Prot_Ms were resuspended in SN_NMR they exhibited elevated activity and acquired inhibition resistance (p<0.05), whereas Prot_NMR resuspended in SN_Ms showed both lower activity and greater sensitivity to inhibition (p<0.05) (means ± S.E.M.; n =5). Bar G reveals that the SN alone had very low peptidolytic activity. (B) Human 26S proteasomes [h26S] treated with SN_NMR, but not SN_Ms or buffer, showed increased ChT-L activity (solid bars) and pronounced resistance to 10nM BZ (hatched bars) (* to *’, p<0.003; ** to **’, p<0.0004; means ± S.E.M; n = 6). (C) SN_NMR conveyed inhibition resistance to the yeast proteasome [y20S]. SN_MS did not show this effect. No significant increase in activity in the presence of SN_NMR was detected. (* and ** indicated p < 0.01; means ± S.E.M.; n = 3)