Figure 3: Initiation length requirements for proteasomal degradation in model proteins.
From: Rad23 escapes degradation because it lacks a proteasome initiation region
The model substrates with terminal initiation regions contained a UbL or Ub4 domain attached to a DHFR domain followed by unstructured regions of different lengths at the C terminus. The substrates with internal initiation regions contained a UbL or Ub4 domain attached to different length unstructured regions followed by a DHFR domain. Degradation experiments were performed at 30 °C. The amount of protein remaining over time was plotted as a percentage of the initial total protein as described in the Methods section. For all types of constructs degradation rates increased with the length of the initiation region. (a) Degradation reactions of UbL-containing substrates with C-terminal initiation regions. The tails consisted of 0 (black circles), 23 (purple squares), 44 (grey triangles), 64 (light blue triangles) and 102 (navy diamonds) amino acids derived from S. cerevisiae cytochrome b2. (b) Degradation reactions of UbL-containing substrates with internal initiation regions, which consisted of 0 (black circles) 60 (light blue triangles), 102 (navy diamonds), 137 (brown triangles), 157 (pink diamonds) and 193 (purple squares) amino acids derived from S. cerevisiae cytochrome b2. (c) Initial degradation rates were plotted against the length of the initiation region given as the number of amino acids (aa). Considerably longer initiation regions are required to support robust degradation when these regions are flanked by folded domains than when they are placed at the C terminus of the substrates. The requirement for long initiation regions does not appear to depend on the details of the amino acid sequence of the initiation region as scrambling the amino acid sequence of the linkers used (black dotted line) did not change the initiation region length dependence of degradation significantly. (d) Degradation reactions of ubiquitin-tagged substrates with C-terminal initiation regions; initiation regions were the same as in a. (e) Degradation reactions of ubiquitin-tagged substrates with internal initiation regions; initiation regions are the same as in b. (f) Initial degradation rates of ubiquitin-tagged substrates plotted against the length of the initiation region given as the number of amino acids. Data points represent mean values and error bars show standard errors calculated from at least three repeat experiments.
