An E2 enzyme Ubc11 is required for ubiquitination of Slp1/Cdc20 and spindle checkpoint silencing in fission yeast

Abstract

For ordered mitotic progression, various proteins have to be regulated by an ubiquitin ligase, the anaphase-promoting complex or cyclosome (APC/C) with appropriate timing. Recent studies have implied that the activity of APC/C also contributes to release of mitotic checkpoint complexes (MCCs) from its target Cdc20 in the process of silencing the spindle assembly checkpoint (SAC). Here we describe a temperature-sensitive mutant (ubc11-P93L) in which cell cycle progression is arrested at mitosis. The mutant grows normally at the restrictive temperature when SAC is inactivated, suggesting that the arrest is not due to abnormal spindle assembly, but rather due to prolonged activation of SAC. Supporting this notion, MCCs remain bound to APC/C even when SAC is satisfied. The ubc11 (+) gene encodes one of the two E2 enzymes required for progression through mitosis in fission yeast. Remarkably, Slp1 (a fission yeast homolog of Cdc20), which is degraded in an APC/C-dependent manner, stays stable throughout the cell cycle in the ubc11-P93L mutant lacking the functional SAC. Other APC/C substrates, in contrast, were degraded on schedule. We have also found that a loss of Ubc4, the other E2 required for progression through mitosis, does not affect the stability of Slp1. We propose that each of the two E2 enzymes is responsible for collaborating with APC/C for a specific set of substrates, and that Ubc11 is responsible for regulating Slp1 with APC/C for silencing the SAC.

Keywords: APC/C; Mad2; SAC; Slp1; Ubc11; mitosis; ubiquitin.

Figure 1. Isolation of the ubc11-P93L mutant. (A) Full-length amino acid sequences of human UbcH10/UBE2C, mouse Ube2C, frog UBCx, clam E2-C, fruitfly Vihar and fission yeast Ubc11 are aligned. A closed circle indicates the mutation site of Ubc11^P93L, where prorine is replaced with leucine. A cysteine residue in UBC domain for thiolester formation with ubiquitin is marked by an asterisk. (B) A wild-type strain, a strain deleted for mad2⁺, Δmad2, slp1-mr63, ubc11-P93L, ubc11-P93L Δmad2, ubc11-P93L slp1-mr63 were spotted on YEA media and were incubated at 26°C or 36°C for 3 d.

Figure 2. Characterization of Ubc11^P93L protein. (A) The reaction mixtures for the in vitro ubiquitin transfer assay were run on SDS-PAGE and silver-stained (left panel) or dried and exposed to the X-ray film (right panel). (B and C) Western blot was performed with extracts prepared from cells incubated at 26°C or 36°C for 4 h. The genotype of each strain is indicated on the top.

Figure 3. Mitotic arrest in the ubc11-P93L mutant. (A) The frequency of respective phenotypes of nuclear morphology: interphase-like normal phenotype, condensed chromosomes, the cut phenotype and aberrant septation are shown at each time point after the shift to the restrictive temperature. (B) The ubc11-P93L mutant cells were incubated for up to 5 h at the restrictive temperature. The sample taken at 2 h after the shift was processed for indirect immunofluorescent staining with the aniti-tubulin antibody (magenta). DNA was visualized by staining with DAPI. The position of the centromere (Cnp1-GFP, green) was also examined. The scale bar indicates 5 μm. The frequency of the cells with Cnp1-GFP on the spindle was also shown at each time point after the shift to the restrictive temperature. (C) Western blot (INPUT) and immunoprecipitation with the anti-FLAG antibody (IP: α-FLAG) were performed with extracts prepared from the ubc11-P93L cells incubated at 26°C or 36°C for up to 5 h.

Figure 4. Stability of Slp1 in the ubc11-P93L mutant. (A) Western blot was performed with extracts prepared from cells incubated at 26°C or 36°C for up to 5 h. The genotype of each strain is indicated on the top. (B) Western blot was performed with extracts prepared from cells incubated at 36°C for 10 h. The genotype of each strain is indicated on the top. (C) Each strain was arrested by the shift to the restrictive temperature for the nda3-KM311 mutation (20°C) for 6 h and released to 36°C at time 0. Cell extracts were prepared at indicated time points and processed for western blotting with each antibody. The genotype of each strain is indicated on the top. Extracts prepared from an asynchronous culture (As) were also examined in the same way.

Figure 5. Ubiquitination of Slp1. (A and B) Cell extracts were prepared from cultures first incubated at 26°C and then shifted up to 36°C for 4 h. Ubiquitinated proteins were purified and analyzed by immunoblotting with the Slp1 antibody. Note that the band likely representing nonubiquitinated Slp1, indicated by asterisk, can also be detected in the pull-down samples, likely due to a His-residue cluster within the Slp1 protein. (C) The cell extracts were prepared and processed as in (A). (D) The nda3-KM311 mutant cells were first incubated at 20°C for 6 h. Cycloheximide was added to the media at time 0 and continuously incubated for up to 60 min. Cell extracts were processed for immunoblot at each time point.