Figure 5.

Spacing requirements for the two degron parts as initiation region length varies. To determine how the spacing of the proteasome-binding tag and initiation region modulates the effect of initiation region length on degradation, we varied proteasome-binding tag - initiation region spacing and initiation region length systematically as shown for constructs with UbL tags (a), Ub₄-tagged substrates were constructed analogously. (b) Degradation kinetics for substrates in which three I27 domains separate a Ub₄ tag from a 44 amino acid (green squares) or a 102 amino acid (light blue squares) initiation region. (c,d) Results for the degradation of other proteins in the array of constructs are shown as plots of initial degradation rates against initiation region lengths for constructs with UbL (c) and Ub₄ (d) tags and zero (red solid diamonds), two (black solid circles), or four (blue solid triangles) I27 domain insertions. Degradation rates increase with the length of the initiation region for both tags but the strength of the response depends on the spacing. (e,f) Plots of initial degradation rates as a function of the distance between the proteasome-binding tag and initiation region for substrates with UbL (e) and Ub₄ (f) tags with 34 amino acid (green solid diamonds), 44 amino acid (orange solid circles), or 102 amino acid (purple solid triangles) initiation regions. The longest initiation regions dampen the effect of degron spacing on degradation rates. Data points represent mean values and error bars show standard errors calculated from three to five repeat experiments.