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1.
FIGURE 3.

FIGURE 3. From: Ubiquitin Ligase Ufd2 Is Required for Efficient Degradation of Mps1 Kinase.

Ufd2 is not required for degradation of all APC substrates. A and B, involvement of Ufd2 in the degradation of APC substrates. Shown are the results from immunoblot analysis of endogenously expressed, C-terminally TAP-tagged Mps1, Ase1, or Clb2 in wild-type and ufd2Δ cultures synchronized with α-factor. Shown is the time after removal of α-factor in wild-type and ufd2Δ cultures. C and D, degradation of two APC substrates (Mad3 and Dbf4) in wild-type or ufd2Δ cells. Yeast cells containing GAL1-regulated Mad3 or Dbf4 tagged with HA and the IgG-binding site from protein A were synchronized in G1 by α-factor arrest and induced with galactose for 2 h. Induction was terminated by the addition of 2% glucose and 100 μg/ml cycloheximide. Samples were withdrawn at intervals and processed for immunoblotting with anti-HA antibody.

Chang Liu, et al. J Biol Chem. 2011 December 23;286(51):43660-43667.
2.
FIGURE 2.

FIGURE 2. From: Ubiquitin Ligase Ufd2 Is Required for Efficient Degradation of Mps1 Kinase.

Involvement of Ufd2 and APC pathway in Mps1 degradation. A, overexpression of various Mps1 alleles causes toxicity in cdc16-1 and ufd2Δ mutants. The plasmid bearing GST-His6-Mps1 or Mps1-Myc or the empty vector was transformed into the strains indicated. 5-Fold serial dilutions were plated as described in the legend to Fig. 1A. B, Mps1-Myc degradation involves the Ufd2-Rad23/Dsk2 pathway. Mps1-Myc stability in the indicated strains was determined as described in the legend to Fig. 1B except that immunoprecipitation was done with Myc beads, followed by immunoblotting with anti-Myc antibody. C, Ufd2 regulates Mps1 ubiquitylation. Myc-tagged Mps1 was transformed with HA-tagged Ub into wild-type or ufd2Δ cells. Because Mps1-Myc ubiquitylation was difficult to detect under normal conditions, we treated wild-type or ufd2Δ cells with the proteasome inhibitor MG132, followed by immunoprecipitation with Myc beads and blotting with anti-HA antibody for ubiquitylated Mps1 species (upper panel). The amounts of Mps1 and Rpt5 in the extracts were also determined by immunoblotting (middle and lower panels).

Chang Liu, et al. J Biol Chem. 2011 December 23;286(51):43660-43667.
3.
FIGURE 4.

FIGURE 4. From: Ubiquitin Ligase Ufd2 Is Required for Efficient Degradation of Mps1 Kinase.

Cell cycle and spindle-related phenotypes associated with ufd2Δ cells. A, the ufd2Δ mutant strains are sensitive to the microtubule-destabilizing drug benomyl. Yeast strains were grown to similar densities, and 7-fold serial dilutions were spotted onto YPD plates with or without benomyl (15 μg/ml). B, deletion of UFD2 suppresses slow growth of bub1Δ, tid3-1, bub3Δ, and spc110-221 mutant cells. Yeast cells with the indicated genotypes were spotted onto synthetic complete plates in serial 10-fold dilutions and incubated at 30 °C for 2 days. C, enhanced chromosome stability in yeast cells lacking UFD2. Sectoring assay for the chromosome stability was carried out as described previously (13). The experiments were done five times, and the means ± S.D. are shown. D, representative images from the sectoring assay for the chromosome loss using the SUP11-containing YPH278 and ufd2Δ mutants. Colonies containing red color or sectors are marked with arrows.

Chang Liu, et al. J Biol Chem. 2011 December 23;286(51):43660-43667.
4.
FIGURE 5.

FIGURE 5. From: Ubiquitin Ligase Ufd2 Is Required for Efficient Degradation of Mps1 Kinase.

Mps1 overexpression-induced phenotypes. A, accumulation of large amounts of Mps1 in ufd2Δ cells delays cell cycle progression. Wild-type or ufd2Δ cells were arrested in G2/M by nocodazole (Noc) for 4 h. Cells were released from the arrest after the removal of nocodazole, and samples taken at various time points after the release were subject to FACScan analysis of DNA content. In the two lower panels, Mps1-Myc expression was induced for 2 h with the addition of galactose and was repressed with glucose as cells were released from the G2/M block. Representative FACS results are shown. OE, overexpression. B, plating efficiency of wild-type or ufd2Δ mutant cells that transiently overexpressed Mps1. Mid-log phase yeast cells were split in two sets: one set was kept unperturbed, the second set was treated with galactose for 1.5 or 3 h to transiently induce Mps1 expression. Equal numbers of cells were plated on glucose-containing medium to repress Mps1 overexpression. The percentage of yeast cells that formed colonies after Mps1 induction was determined. Values shown are derived from three independent experiments.

Chang Liu, et al. J Biol Chem. 2011 December 23;286(51):43660-43667.
5.
FIGURE 6.

FIGURE 6. From: Ubiquitin Ligase Ufd2 Is Required for Efficient Degradation of Mps1 Kinase.

UFD2a regulates human MPS1 degradation. A, human MPS1 degradation is compromised in UFD2a-deficient cells. Wild-type UFD2a-expressing SH-SY5Y cells and UFD2a-deficient NB-1 cells were transiently transfected with Myc-tagged MPS1. 24 h after transfection, cells were split onto a 6-well plate. After 48 h of transfection, cells were treated with 100 μg/ml cycloheximide (CHX) to start the chase. Samples were taken at the indicated time points after protein synthesis shut-off and analyzed by Western blotting with anti-Myc antibody. B, UFD2a expression restores MPS1 turnover in NB-1 cells. The plasmid expressing FLAG-tagged UFD2a or the vector plasmid was transfected into NB-1 cells bearing Myc-tagged MPS1. MPS1 degradation was assessed as described for A. UFD2a expression was ascertained by Western blotting with anti-FLAG antibody. C, MPS1 binds endogenous UFD2a by co-immunoprecipitation. UFD2a-deficient NB-1 cells and UFD2a-containing SH-SY5Y cells were transiently transfected with Myc-tagged human MPS1 or vector as indicated. After 48 h of transfection, cell extracts were prepared and subjected to immunoprecipitation (IP) with anti-Myc antibody, followed by immunoblotting with anti-UFD2a antibody. In the lower panels, 10% input lysates were analyzed by immunoblotting to ascertain the expression of UFD2a and Myc-MPS1. In the right panels, the MPS1-UFD2a interaction was determined in Cdc27 siRNA (siCdc27)- or mock-treated SH-SY5Y cells. Cdc27 knockdown efficiency is shown in the lower right panel. D, UFD2a affects MPS1-Cdc27 association. The binding experiments were carried out as described for C using FLAG-tagged UFD2a and Myc-tagged MPS1 in SH-SY5Y or NB-1 cells. E, UFD2a interacts with Cdc27. The immunoprecipitations were done as described for C. Endogenous Cdc27 was detected using anti-Cdc27 antibody.

Chang Liu, et al. J Biol Chem. 2011 December 23;286(51):43660-43667.
6.
FIGURE 1.

FIGURE 1. From: Ubiquitin Ligase Ufd2 Is Required for Efficient Degradation of Mps1 Kinase.

Identification of Mps1 as substrate of Ufd2-Rad23/Dsk2 pathway. A, overexpression of Mps1 leads to slower growth of ufd2Δ or rad23Δ dsk2Δ double mutant cells. GST-His6-Mps1 isolated from the genome-wide screen was transformed into the wild type or the indicated mutants. These cells were grown to similar densities, and 5-fold serial dilutions were spotted onto SD or SG medium. B, efficient degradation of Mps1 requires Ufd2, Rad23, and Dsk2. Wild-type and ufd2Δ cells containing GAL1 promoter-driven GST-His6-Mps1 were first grown in raffinose-containing medium. Expression of Mps1 was induced by the addition of galactose. Samples were taken after promoter shut-off at the time points indicated and analyzed by anti-His6 Western blotting. Equal amounts of protein extracts were used and confirmed by blotting with anti-Rpt5 antibody in all of the expression shut-off experiments (lower panel). Proteins are identified on the left. C, Mps1 is phosphorylated. GST-His6-Mps1 was expressed in wild-type cells and recovered by immunoprecipitation. Mps1 immunoprecipitates were incubated with or without alkaline phosphatase and visualized by immunoblotting. CIP, calf intestinal phosphatase. D, the U-box motif is critical for Mps1 degradation. GST-tagged Mps1 was transformed into the wild type and ufd2U-boxΔ mutants. Mps1 degradation was assayed as described for B. E and F, Mps1 degradation requires Ufd1 but not Rpn10. GST-His6-Mps1 stabilities in ufd1-1 and rpn10Δ were determined.

Chang Liu, et al. J Biol Chem. 2011 December 23;286(51):43660-43667.

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