Bioinformatics analysis identifies several intrinsically disordered human E3 ubiquitin-protein ligases

The ubiquitin-proteasome system targets misfolded proteins for degradation. Since the accumulation of such proteins is potentially harmful for the cell, their prompt removal is important. E3 ubiquitin-protein ligases mediate substrate ubiquitination by bringing together the substrate with an E2 ubiquitin-conjugating enzyme, which transfers ubiquitin to the substrate. For misfolded proteins, substrate recognition is generally delegated to molecular chaperones that subsequently interact with specific E3 ligases. An important exception is San1, a yeast E3 ligase. San1 harbors extensive regions of intrinsic disorder, which provide both conformational flexibility and sites for direct recognition of misfolded targets of vastly different conformations. So far, no mammalian ortholog of San1 is known, nor is it clear whether other E3 ligases utilize disordered regions for substrate recognition. Here, we conduct a bioinformatics analysis to examine >600 human and S. cerevisiae E3 ligases to identify enzymes that are similar to San1 in terms of function and/or mechanism of substrate recognition. An initial sequence-based database search was found to detect candidates primarily based on the homology of their ordered regions, and did not capture the unique disorder patterns that encode the functional mechanism of San1. However, by searching specifically for key features of the San1 sequence, such as long regions of intrinsic disorder embedded with short stretches predicted to be suitable for substrate interaction, we identified several E3 ligases with these characteristics. Our initial analysis revealed that another remarkable trait of San1 is shared with several candidate E3 ligases: long stretches of complete lysine suppression, which in San1 limits auto-ubiquitination. We encode these characteristic features into a San1 similarity-score, and present a set of proteins that are plausible candidates as San1 counterparts in humans. In conclusion, our work indicates that San1 is not a unique case, and that several other yeast and human E3 ligases have sequence properties that may allow them to recognize substrates by a similar mechanism as San1

A Chaperone-Assisted Degradation Pathway Targets Kinetochore Proteins to Ensure Genome Stability

<div><p>Cells are regularly exposed to stress conditions that may lead to protein misfolding. To cope with this challenge, molecular chaperones selectively target structurally perturbed proteins for degradation via the ubiquitin-proteasome pathway. In mammals the co-chaperone BAG-1 plays an important role in this system. BAG-1 has two orthologues, Bag101 and Bag102, in the fission yeast <i>Schizosaccharomyces pombe</i>. We show that both Bag101 and Bag102 interact with 26S proteasomes and Hsp70. By epistasis mapping we identify a mutant in the conserved kinetochore component Spc7 (Spc105/Blinkin) as a target for a quality control system that also involves, Hsp70, Bag102, the 26S proteasome, Ubc4 and the ubiquitin-ligases Ubr11 and San1. Accordingly, chromosome missegregation of <i>spc7</i> mutant strains is alleviated by mutation of components in this pathway. In addition, we isolated a dominant negative version of the deubiquitylating enzyme, Ubp3, as a suppressor of the <i>spc7-23</i> phenotype, suggesting that the proteasome-associated Ubp3 is required for this degradation system. Finally, our data suggest that the identified pathway is also involved in quality control of other kinetochore components and therefore likely to be a common degradation mechanism to ensure nuclear protein homeostasis and genome integrity.</p></div

Other kinetochore mutants are also suppressed by <i>ubr11</i>Δ and <i>san1</i>Δ.

<p>(A) The growth on solid media of wild type, <i>ubr11</i>Δ, <i>mis6-302</i> and the <i>ubr11</i>Δ<i>mis6-302</i> double mutant was compared at the indicated temperatures. (B) The growth on solid media of wild type, <i>ubr11</i>Δ, <i>mal2-1</i> and the <i>ubr11</i>Δ<i>mal2-1</i> double mutant was compared at the indicated temperatures. (C) The growth on solid media of wild type, <i>san1</i>Δ, <i>mis6-302</i> and the <i>san1</i>Δ<i>mis6-302</i> double mutant was compared at the indicated temperatures. (D) The growth on solid media of wild type, <i>san1</i>Δ, <i>mal2-1</i> and the <i>san1</i>Δ<i>mal2-1</i> double mutant was compared at the indicated temperatures. (E) The growth on solid media of wild type, <i>ubc4-1</i>, <i>mis6-302</i> and the <i>ubc4-1mis6-302</i> double mutant was compared at the indicated temperatures. (F) The growth on solid media of wild type, <i>ubc4-1</i>, <i>mal2-1</i> and the <i>ubc4-1mal2-1</i> double mutant was compared at the indicated temperatures. (G) The growth on solid media of wild type, <i>bag102</i>Δ, <i>mis6-302</i> and the <i>bag102</i>Δ<i>mis6-302</i> double mutant was compared at the indicated temperatures. (H) The growth on solid media of wild type, <i>bag102</i>Δ, <i>mal2-1</i> and the <i>bag102</i>Δ<i>mal2-1</i> double mutant was compared at the indicated temperatures.</p

Spc7-23 is degraded via the ubiquitin-proteasome pathway.

<p>(A) The growth on solid media of wild type (lower panel) and <i>spc7-23</i> cells (upper panel) transformed with either a control vector (vector) or PstI digested genomic DNA construct encoding <i>mts2-219X</i> was compared at different temperatures. (B) The growth of wild type, <i>mts2-1</i>, <i>spc7-23</i> and the <i>mts2-1spc7-23</i> double mutant was compared at the indicated temperatures. (C) The growth on solid media of wild type, <i>nas6</i>Δ, <i>spc7-23</i> and the <i>nas6</i>Δ<i>spc7-23</i> double mutant was compared at the indicated temperatures. (D) Equal (wild type) or unequal DNA segregation was quantified in <i>spc7-23-gfp</i> and <i>spc7-23-gfp nas6</i>Δ cells at 30°C. ** p<0.01 (Welch test) for the <i>spc7-23-gfp nas6</i>Δ strain compared to the <i>spc7-23-gfp</i> strain. For <i>spc7-23-gfp</i> (n = 200) and <i>spc7-23-gfp nas6</i>Δ (n = 100) late anaphase cells. Note that wild type DNA segregation is re-established in the <i>spc7-23nas6</i>Δ double mutant. (E) The amount of Spc7-23 protein was followed in cultures at 27°C and 30°C where protein synthesis was inhibited with 100 µg/mL cycloheximide (CHX) for 4 hours. To some cultures 1 mM of the proteasome inhibitor Bortezomib (BZ) was also added. Equal loading was checked using antibodies to tubulin. (F) The growth on solid media of wild type and <i>spc7-23</i> cells was compared at the indicated temperatures in the absence (control) or presence of 100 µM BZ. (G) Strains with the indicated genetic backgrounds and transformed to express 6His-tagged ubiquitin were lysed and used for precipitation experiments with a Ni²⁺ resin in 8 M urea. The precipitated material was analyzed by blotting with antibodies to the HA-tag on Spc7-23 or to the 6His tag on ubiquitin. The arrowhead marks the position where non-ubiquitylated Spc7-23 migrates. Note that ubiquitylated Spc7-23 species are visible in proteasome and <i>bag102</i>Δ mutants.</p

Spc7-23 degradation depends on the proteasome-associated DUB Ubp3.

<p>(A) The growth on solid media of <i>spc7-23</i> cells transformed with either a control plasmid (vector) or expression constructs for <i>ubp3</i>⁺ and <i>ubp3-W466X</i> was compared at different temperatures. (B) The growth on solid media of wild type, <i>ubp3</i>Δ, <i>spc7-23</i> and the <i>ubp3</i>Δ<i>spc7-23</i> double mutant was compared at the indicated temperatures. (C) Equal (wild type) or unequal DNA segregation was quantified in <i>spc7-23-gfp</i> and <i>spc7-23-gfp ubp3</i>Δ cells at 30°C. ** p<0.01 (Welch test) for the <i>spc7-23-gfp ubp3</i>Δ strain compared to the <i>spc7-23-gfp</i> strain. For <i>spc7-23-gfp</i> (n = 200) and <i>spc7-23-gfp ubp3</i>Δ (n = 100) late anaphase cells. Note that wild type DNA segregation is re-established in the <i>spc7-23ubp3</i>Δ double mutant. (D) Cells transformed with vector (control) or Ubp3-Flag were used for immunoprecipitation experiments using antibodies to the Flag epitope. The precipitated material was analyzed by blotting for the proteasome subunit Mts4 (upper panel) or as a loading control Ubp3 (lower panel). (E) Wild type or <i>ubp3</i>Δ cells transformed to express 6His-tagged ubiquitin, were lysed and used for precipitation experiments with a Ni²⁺ resin in 8 M urea. The precipitated material was analyzed by blotting with antibodies to the HA-tag on Spc7-23 or the 6His tag on ubiquitin. The arrowhead marks the position where non-ubiquitylated Spc7-23 migrates.</p

A chaperone-assisted degradation pathway for nuclear proteins.

<p>The data presented here are compatible with a model where a nuclear protein becomes structurally perturbed to a degree where it is still functional, but molecular chaperones detect it as being misfolded. The protein is then ubiquitylated by the E2 and E3 enzymes Ubc4, Ubr11 and San1, and directed to the 26S proteasome via Bag102. Finally, at the 26S proteasome, the protein is deubiquitylated by the DUB Ubp3 and degraded. Ubiquitin is shown as grey spheres. The structural perturbation is shown as a star.</p

Spc7-23 degradation depends on Ubc4, Ubr11 and San1.

<p>(A) The growth of the indicated strains on solid media was compared at 25°C (upper panel) and 30°C (lower panel). (B) The growth of the indicated strains on solid media was compared at 25°C (left panel) and 30°C (right panel). (C) Equal (wild type) or unequal DNA segregation was quantified in <i>spc7-23-gfp ubr11</i>Δ and <i>spc7-23-gfp san1</i>Δ cells at 30°C. ** p<0.01 (Welch test) for the <i>spc7-23-gfp ubr11</i>Δ and <i>spc7-23-gfp san1</i>Δ strains compared to the <i>spc7-23-gfp</i> strain. For <i>spc7-23-gfp</i> (n = 200), <i>spc7-23-gfp ubr11</i>Δ (n = 100) and <i>spc7-23-gfp san1</i>Δ (n = 100) late anaphase cells. Note that wild type DNA segregation is re-established in the <i>spc7-23ubr11</i>Δ and <i>spc7-23san1</i>Δ double mutants. (D) V5-tagged Ubr11 was immunoprecipitated from cells treated with Bortezomib (BZ) using antibodies to V5 or control antibody. The precipitated material was analyzed by SDS-PAGE and blotting for V5 (Ubr11) and HA (Spc7). (E) Flag-tagged San1 was immunoprecipitated from cells treated with Bortezomib (BZ) using antibodies to Flag or control antibody. The precipitated material was analyzed by SDS-PAGE and blotting for Flag (San1) and HA (Spc7). (F) Strains with the indicated genetic backgrounds and transformed to express 6His-tagged ubiquitin were either not treated or treated with 1 mM of the proteasome inhibitor Bortezomib (BZ) overnight. 6His-tagged ubiquitin was precipitated with a Ni²⁺ resin in 8 M urea. The precipitated material was analyzed by SDS-PAGE and blotting with antibodies to the HA-tag on Spc7-23. The arrowhead marks the position where non-ubiquitylated Spc7-23 migrates. Note that ubiquitylated Spc7-23 species are less abundant in <i>ubr11</i>Δ and <i>san1</i>Δ mutants.</p

<i>spc7-23</i> cells are temperature sensitive and defective in DNA segregation.

<p>(A) The growth of wild type and <i>spc7-23</i> cells on solid media was compared at the indicated temperatures. (B) Cells carrying the mutant <i>spc7-23</i> mutation were incubated at 25°C or 30°C and stained to visualize DNA (blue) and tubulin (green). Note that the DNA segregation becomes unequal at the restrictive temperature.</p

Bag101 and Bag102 interact with 26S proteasomes and Hsp70.

<p>(A) Domain organization (shown to scale) of human BAG-1S and the <i>S. pombe</i> homologs Bag101 and Bag102. The truncations used for the precipitation experiments are shown. (B) The indicated GST fusion proteins were used in pull down experiments with extract from <i>S. pombe</i> cells expressing ZZ-tagged Rpn11. The precipitated material was analyzed by SDS-PAGE and blotting for Hsp70 (upper panel), the ZZ-tagged 26S proteasome subunit Rpn11, and 20S particle α subunits (middle panels). Equal loading was checked by staining with Coomassie Brilliant Blue (CBB) (lower panel). (C) Immunoprecipitates from wild type <i>S. pombe</i> cells expressing Flag-tagged Bag101 and Bag102 were resolved by SDS-PAGE and analyzed by blotting, using antibodies to the proteasome subunit Mts4/Rpn1, and Flag. (D) Differential interference contrast (DIC) and fluorescence micrographs of wild type <i>S. pombe</i> transformed to express GFP-tagged Bag101 and Bag102. DAPI staining was used to mark the nucleus. (E) Lysates from <i>S. pombe</i> cells transformed to express Flag- and GFP-tagged Bag102 were separated into a soluble fraction and pellet. The pellet fraction was then treated with proteinase K and Triton X-100 as indicated, before the samples were analyzed by SDS-PAGE and blotting. Dph1 served as a control for a soluble protein. Bip1 served as a control for an ER luminal protein.</p