Figure 1: Rad23 escapes proteasome degradation in vitro because it lacks an initiation region.

The amount of protein remaining over time was plotted as a percentage of the initial total protein as described in the Methods section. (a) Degradation kinetics of Rad23. Incubation of Rad23 with purified yeast proteasome did not lead to its degradation at 30 °C. (b) The stability of Rad23 was not due to the UBA2 domain. Both UBA domains were replaced by dihydrofolate reductase (DHFR) individually or in combination and none of the substrates were degraded by the proteasome. Appending a 95 amino-acid unstructured region from S. cerevisiae cytochrome b₂ to the C terminus of any of the Rad23 hybrid proteins in which either or both of the UBA domains had been replaced with a DHFR domain led to its rapid degradation. (c) Adding an initiation region to Rad23 led to its rapid degradation. Unstructured tails of different lengths were placed at the C terminus of Rad23 to serve as initiation regions. Rad23 with either a 39 amino-acid unstructured region from the E. coli lac repressor (pink squares) or a 95 amino-acid unstructured region from S. cerevisiae cytochrome b₂ (blue diamonds) added as an initiation region led to its degradation. Deletion of the UBA2, leaving a 45 amino-acid unstructured region, led to Rad23's degradation (light blue circles). (d) Degradation of Rad23 with a 95 amino-acid initiation region at its C terminus was proteasome dependent. Rad23-95 was stable when the proteasome was inhibited with MG132 (purple squares), in the absence of ATP (brown diamonds) in the degradation reaction, when its UbL domain was deleted (black circles). Rad23-95 was otherwise rapidly degraded by the proteasome (navy diamonds). Data points represent mean values and were calculated from at least three repeat experiments.