The Mub1/Ubr2 ubiquitin ligase complex regulates the conserved Dsn1 kinetochore protein

Abstract

The kinetochore is the macromolecular complex that assembles onto centromeric DNA and orchestrates the segregation of duplicated chromosomes. More than 60 components make up the budding yeast kinetochore, including inner kinetochore proteins that bind to centromeric chromatin and outer proteins that directly interact with microtubules. However, little is known about how these components assemble into a functional kinetochore and whether there are quality control mechanisms that monitor kinetochore integrity. We previously developed a method to isolate kinetochore particles via purification of the conserved Dsn1 kinetochore protein. We find that the Mub1/Ubr2 ubiquitin ligase complex associates with kinetochore particles through the CENP-C(Mif2) protein. Although Mub1/Ubr2 are not stable kinetochore components in vivo, they regulate the levels of the conserved outer kinetochore protein Dsn1 via ubiquitylation. Strikingly, a deletion of Mub1/Ubr2 restores the levels and viability of a mutant Dsn1 protein, reminiscent of quality control systems that target aberrant proteins for degradation. Consistent with this, Mub1/Ubr2 help to maintain viability when kinetochores are defective. Together, our data identify a previously unknown regulatory mechanism for the conserved Dsn1 kinetochore protein. We propose that Mub1/Ubr2 are part of a quality control system that monitors kinetochore integrity, thus ensuring genomic stability.

Figure 1. Dsn1 associates with an E3 ubiquitin ligase, Ubr2, via its adaptor, Mub1.

(A) Dsn1 associates with Ubr2. Dsn1-His-Flag was purified from cells containing either pGAL-GFP-UBR2 (SBY8605) or UBR2 (SBY8253) after 2 hours of growth in galactose and analyzed via silver staining. The prominent band migrating ∼190 kDa is shifted up when GFP-Ubr2 is expressed, confirming that it is Ubr2 (Ubr2: 217 kDa, GFP: 27 kDa). Note that Mub1 runs at the same position as background bands (indicated by asterisk). (B) Co-immunoprecipitation confirms that Dsn1 associates with Mub1. Proteins were purified with anti-Flag antibodies from cells containing Mub1-Myc that express either Dsn1-His-Flag (SBY8550) or untagged Dsn1 (SBY8590) and analyzed by immunoblot. Note that the Dsn1-His-Flag band overlaps with a background signal in the input. (C) The association between Dsn1 and Ubr2 requires Mub1. Dsn1-His-Flag was purified from cells in the presence (SBY8253) or absence (SBY8480) of MUB1. The band corresponding to Ubr2 is absent in mub1Δ cells. Background bands are indicated by asterisk.

Figure 2. CENP-C recruits Mub1/Ubr2 onto Dsn1-derived kinetochore particles.

(A) Dsn1 association with Ubr2 requires CENP-C^Mif2. Dsn1-His-Flag was immunoprecipitated from cells with either MIF2 (SBY8253) or mif2-3 (SBY8405) and analyzed via SDS-PAGE and silver staining. Note that CENP-C^Mif2 runs at the same position as background bands (indicated by asterisk). (B) Dsn1 association with Mub1 requires CENP-C^Mif2. Dsn1-His-Flag was immunoprecipitated from cells containing Mub1-Myc and either MIF2 (SBY8550) or mif2-3 (SBY8551). (C) CENP-C^Mif2 associates with Ubr2. Dsn1-His-Flag (SBY8253) and CENP-C^Mif2-Flag (SBY8519) were immunoprecipitated and analyzed via SDS-PAGE and silver staining. (D) CENP-C^Mif2-Flag MS summary table. See Table S1 for all proteins identified by MS. (E) CENP-C^Mif2 association with Ubr2 requires Mub1. CENP-C^Mif2-Flag was immunoprecipitated from cells in the presence (SBY8519) or absence (SBY8911) of MUB1 and analyzed via SDS-PAGE and silver staining. (F) The association between CENP-C^Mif2 and Mub1 does not require Ubr2. CENP-C^Mif2-Flag was immunoprecipitated from cells containing Mub1-Myc in the presence (SBY8546) or absence (SBY8572) of UBR2 and analyzed via immunoblot. The Mub1 protein level in the input is higher in ubr2Δ due to the lack of Ubr2-dependent proteolysis . Note that the CENP-C^Mif2-Flag band overlaps with a background signal in the input.

Figure 3. Dsn1-S240A,S250A levels correlate with viability.

(A) Dsn1-S240A,S250A cells are inviable. Serial dilutions (3-fold) of dsn1Δ cells containing DSN1 on a URA3, CEN vector and the indicated integrated point mutants (SBY2318, SBY5948, SBY5949, SBY5950) were plated. Cells that need to maintain the URA3, CEN vector for viability are sensitive to 5-FOA. (B) Dsn1-S240A,S250A protein levels are reduced. Whole cell extracts of Dsn1 and the indicated point mutants (SBY2153, SBY7864, SBY7865, SBY7867) were prepared and analyzed via immunoblot. (C) Overexpression of Dsn1-S240A,S250A restores protein levels. Whole cell extracts of the indicated Dsn1 mutants (SBY8766, SBY8521, SBY7373) were prepared and analyzed via immunoblot. (D) Overexpression of dsn1-S240A,S250A restores viability. Serial dilutions (3-fold) of dsn1Δ strains containing DSN1 on a URA3, CEN vector and a 2 µm LEU2 plasmid with the indicated point mutants (SBY7368, SBY7362, SBY7363, SBY7364 were plated on –ura leu and 5-FOA plates. We did not detect any obvious defect in cells overexpressing wild-type or mutant Dsn1 proteins.

Figure 4. Mub1/Ubr2 mediate Dsn1 ubiquitylation and regulate protein levels.

(A) Deleting MUB1 and UBR2 restores Dsn1-S240A,S250A protein levels. Whole cell extracts were prepared from WT (SBY8766, SBY8521), mub1Δ (SBY10959, SBY8164), and ubr2Δ cells (SBY10960, SBY8265). Dsn1-Myc and Dsn1-S240,S250A-Myc levels were monitored by immunoblot. (B) Mub1 regulates Dsn1 stability. WT (SBY8766) and mub1Δ (SBY10959) cells containing Dsn1-myc were treated with cycloheximide and analyzed for Dsn1 protein levels at the indicated time points (min). (C) Aurora B regulates Dsn1 stability. WT (SBY8766) and ipl1-321 (SBY8150) cells containing Dsn1-myc were shifted to 37°C and treated with cycloheximide. Cells were analyzed for Dsn1 protein levels at the indicated time points (min). (D) Mub1/Ubr2 regulate Dsn1 stability in an Aurora B mutant. Ipl1-321 (SBY8150) and ipl1-321 mub1Δ (SBY9428) cells containing Dsn1-myc were shifted to 37°C and treated with cycloheximide. Cells were analyzed for Dsn1 protein levels at the indicated time points (min). (E) Ubr2 ubiquitylates Dsn1-S240A,S250A and wild-type Dsn1. Flag epitope-tagged Dsn1-S240A,S250A or Dsn1-WT was immunoprecipitated from cim3-1 ubr2Δ cells (SBY8703, SBY8705), cim3-1 UBR2 cells (SBY8704, SBY8706) and WT cells (SBY8615, SBY7441). Samples were analyzed via immunoblot with anti-Flag and anti-Ub antibodies. (F) Deleting MUB1 and UBR2 restores viability to Dsn1-S240A,S250A cells. Serial dilutions (3-fold) of pGAL-DSN1 cells containing integrated dsn1-S240A,S250A with the indicated deletions (SBY8264, SBY8262, SBY8844, SBY8469, SBY8842, SBY8901) were plated on either glucose or galactose media. A WT strain (SBY3) and a pGAL-DSN1 strain lacking the integrated point-mutant (SBY7948) were included as controls.

Figure 5. Mub1/Ubr2 cannot be detected at endogenous centromeres but are important for kinetochore function.

(A) Mub1 is dispensable for kinetochore biorientation. Mtw1-3GFP was monitored in either MUB1 (SBY3798) or mub1Δ (SBY8026) cells for biorientation defects. A representative cell from each strain is shown. Bar, 5 µm. (B) Mub1 cannot be detected at centromeres. Chromatin immunoprecipitation was carried-out on Mub1-Myc ubr2Δ (SBY8572), Mif2-Myc (SBY1566), and untagged control (SBY3) cells using a probe for CEN3. We obtained similar results using Mub1-Myc UBR2 cells (data not shown). (C, D) Mub1Δ and ubr2Δ exhibit negative genetic interactions with kinetochore mutants. Serial dilutions (5-fold) of ndc10-1 (SBY3, SBY7793, SBY7851, SBY164, SBY8613, SBY8773) and ndc80-1 (SBY3, SBY7793, SBY7851, SBY1117, SBY8436, SBY8432) kinetochore mutants with mub1Δ and ubr2Δ were plated on YPD and incubated at the indicated temperatures to examine genetic interactions. (E) Ndc80-1 mub1Δ double mutants exhibit an increase in declustered kinetochores. Ndc80-1 (SBY3934) and ndc80-1 mub1Δ (SBY8670) cells containing Mtw1-3GFP were released from G1 to 30 degrees and kinetochores were visualized at 180′. The percent of clustered (left panel) vs. unclustered (right panel) was quantified. Bar, 5 µm.