Figure 5
Shutting down E1 activity in ts20 cells. (a) Time course of conversion of conjugates into monomeric ubiquitin pool Western blot analysis of cell extracts from ts20 cells cultured either at 34°C or 42°C for the indicated time with an antibody recognizing ubiquitin. The analysis revealed a detectable conversion of ubiquitin conjugates into monomeric ubiquitin in cells cultured at 42°C for 2 hours. Maintaining the cells at 42°C for an additional 1 or 2 hours did not significantly enhance the conversion of conjugates into the monomeric pool. The addition of proteasome inhibitor in the culture medium of cells cultured at 42°C blocked the conversion of poly-ubiquitinated conjugates and resulted in their enhanced detection. The membrane was re-probed with an actin specific antibody which served as a loading control (boxed panel). (b) Model output E1 activity was blocked at time = 0.5 hours by setting the parameter k62 = 0 at this time point (indicated by dashed vertical line on the graph). All other parameters as in Tables 2 and 3. The simulations were repeated 100 times and the curves show the mean values. Ub conjugates includes all ubiquitinated misfolded protein both free and bound to the proteasome; total bound Ub includes ubiquitin bound to E1 and E2 enzymes in addition to ubiquitinated misfolded proteins. (c) As (b) except ten-fold lower DUB activity on polyubiquitinated proteasome-bound substrates (k68 = 1.0 × 10-6)