Rad23 escapes degradation because it lacks a proteasome initiation region

Abstract

Rad23 is an adaptor protein that binds to both ubiquitinated substrates and to the proteasome. Despite its association with the proteasome, Rad23 escapes degradation. Here we show that Rad23 remains stable because it lacks an effective initiation region at which the proteasome can engage the protein and unfold it. Rad23 contains several internal, unstructured loops, but these are too short to act as initiation regions. Experiments with model proteins show that internal loops must be surprisingly long to engage the proteasome and support degradation. These length requirements are not specific to Rad23 and reflect a general property of the proteasome.

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 DHFR (dihydrofolate reductase) 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 substrates were stable when the proteasome was inhibited with MG132 (purple squares) and in the absence of ATP (brown diamonds) in the degradation reaction and 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.

Figure 2

Internal loops of Rad23 are too short to serve as effective initiation regions. Degradation kinetics at 30 °C are shown of Rad23 substrates with different linker regions. The amount of protein remaining over time was plotted as a percentage of the initial total protein as described in the methods and degradation kinetics of Rad23 (black circles) and Rad23-95 (navy diamonds) are shown. (a) The amino acid sequences of the linker regions of Rad23 were not responsible for the lack of degradation. Replacement of each linker region with a sequence from S. cerevisiae cytochrome b₂ of the same length resulted in inefficient degradation by the proteasome. (b,c) Initiation regions have to be longer within a protein than at the end of a protein. Insertion of 95 (b) or 190 (c) amino acid long sequences into the Rad23 linker regions led to increasing degradation of the hybrid proteins. Data points represent mean values and were calculated from at least three repeat experiments.

Figure 3

Initiation length requirements for proteasomal degradation in model proteins. 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 23 (purple), 44 (gray), 64 (light blue), and 102 (navy) amino acids derived from S. cerevisiae cytochrome b₂. (b) Degradation reactions of UbL containing substrates with internal initiation regions, which consisted of 60 (light blue), 102 (navy), 137 (brown), 157 (pink), and 193 (purple) 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. 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.

Figure 4

The stability of Rad23 in vivo is also determined by the presence of an initiation region. Rad23 with an N-terminal Flag tag expressed in S. cerevisiae accumulated and protein levels were not affected by MG132 as determined by Western blotting for the Flag tag in yeast extracts. Addition of a 39 amino acid long tail to the C terminus of Rad23 (Rad23-39) or insertion of a 190 amino acids long linker into loop 3 (Rad23-190→L3) destabilized the protein and reduced steady-state levels in the cell. Inhibition of proteasomal degradation with MG132 stabilized the Rad23 hybrids and increased their steady state levels. Rad23 with a 95 amino acid long tail (Rad23-95) could not be detected even when the proteasome was inhibited with MG132 but deletion of the UbL domain stabilized the protein sufficiently to allow it to accumulate. Insertion of a 95 amino acid linker into loop 2 did not destabilize the protein noticeably (Rad23-95→L2). No Flag-tagged Rad23 was detected in lysate of cells transfected with plasmid lacking the Rad23 insert and PGK (phosphoglycerate kinase) was measured as a loading control to allow comparison of protein levels in the different lysates.

Figure 5

Analysis of loops within UbL-UBA proteins. Homologues of Rad23, Dsk2, and Ddi1 were retrieved from species selected broadly from evolution (see supplementary information). The loops within these proteins are defined as sequences that fall between motifs found in the SMART and Pfam databases. (a) Schematic of UbL-UBA proteins. Domains and loops represented are not to scale. In addition to UbL and UBA, abbreviations include ST2 (Sti1/Sti1 pair) and ASP (Asp_protease). Note that RBD (Rad4-binding domain) corresponds to the Pfam domain XPC. (b) Relative conservation of domains and loops. Conservation was evaluated by aligning individual domains with Clustal W, extracting the pairwise percent conservation, and averaging. Domains are represented by horizontal segments. Rad23 is shown in red, Dsk2 Class I and II are shown in dark and light blue, respectively, and Ddi1 is shown in green. To align all of the UBA domains in the schematic, break points are introduced into the traces for Class I Dsk2 and Ddi1. (c) Plot of loop lengths. The color scheme for the loops is continued from (b). Black dots indicate the average loop length.

Figure 6

Schematic representation of the length requirement for internal and terminal initiation regions. Substrates shown contained a UbL domain bound to the proteasome in an arbitrary position attached to either a DHFR (dihydrofolate reductase) domain followed by an initiation region (blue) or an initiation region (red and blue) followed by a DHFR domain. Length requirements for an internal initiation region (red and blue) were at least twice that for a terminal initiation region (blue). Light grey: proteasome caps; dark grey: proteasome core; circular arrows: ATPase subunits; scissors; proteolytic sites.