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

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

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

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

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

<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