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Dma1 ubiquitinates the SIN scaffold, Sid4, to impede the mitotic localization of Plo1 kinase

2010; Springer Nature; Volume: 30; Issue: 2 Linguagem: Inglês

10.1038/emboj.2010.317

1460-2075

Alyssa E. Johnson, Kathleen L. Gould,

Genomics and Chromatin Dynamics

Article3 December 2010free access Dma1 ubiquitinates the SIN scaffold, Sid4, to impede the mitotic localization of Plo1 kinase Alyssa E Johnson Alyssa E Johnson Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN, USA Search for more papers by this author Kathleen L Gould Corresponding Author Kathleen L Gould Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN, USA Howard Hughes Medical Institute, Vanderbilt University School of Medicine, Nashville, TN, USA Search for more papers by this author Alyssa E Johnson Alyssa E Johnson Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN, USA Search for more papers by this author Kathleen L Gould Corresponding Author Kathleen L Gould Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN, USA Howard Hughes Medical Institute, Vanderbilt University School of Medicine, Nashville, TN, USA Search for more papers by this author Author Information Alyssa E Johnson1 and Kathleen L Gould 1,2 1Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN, USA 2Howard Hughes Medical Institute, Vanderbilt University School of Medicine, Nashville, TN, USA *Corresponding author. Department of Cell and Developmental Biology, Howard Hughes Medical Institute and Vanderbilt University, Vanderbilt University School of Medicine, 21st avenue S, Nashville, TN 37232, USA. Tel.: +1 615 343 9500; Fax: +1 615 343 0723; E-mail: [email protected] The EMBO Journal (2011)30:341-354https://doi.org/10.1038/emboj.2010.317 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Proper cell division requires strict coordination between mitotic exit and cytokinesis. In the event of a mitotic error, cytokinesis must be inhibited to ensure equal partitioning of genetic material. In the fission yeast, Schizosaccharomyces pombe, the checkpoint protein and E3 ubiquitin ligase, Dma1, delays cytokinesis by inhibiting the septation initiation network (SIN) when chromosomes are not attached to the mitotic spindle. To elucidate the mechanism by which Dma1 inhibits the SIN, we screened all SIN components as potential Dma1 substrates and found that the SIN scaffold protein, Sid4, is ubiquitinated in vivo in a Dma1-dependent manner. To investigate the role of Sid4 ubiquitination in checkpoint function, a ubiquitination deficient sid4 allele was generated and our data indicate that Sid4 ubiquitination by Dma1 is required to prevent cytokinesis during a mitotic checkpoint arrest. Furthermore, Sid4 ubiquitination delays recruitment of the Polo-like kinase and SIN activator, Plo1, to spindle pole bodies (SPBs), while at the same time prolonging residence of the SIN inhibitor, Byr4, providing a mechanistic link between Dma1 activity and cytokinesis inhibition. Introduction At the end of each cell division cycle, chromosomes segregate to opposite sides of the cell and a cytokinetic ring (CR) assembles and constricts between them to physically separate the two new cells. Clearly, it is critical that chromosome segregation occurs before ring constriction and, thus, mitosis and cytokinesis must be coupled to ensure that each new cell inherits the proper genetic complement. In the fission yeast, Schizosaccharomyces pombe, the septation initiation network (SIN) confers proper coordination by triggering contractile ring constriction once mitosis is complete (for review see McCollum and Gould, 2001; Krapp et al, 2004b). Thus, precise activation of the SIN is required for the fidelity of each cell division. SIN signalling is restricted to the spindle pole bodies (SPBs) and is initiated by the GTPase, Spg1 (Schmidt et al, 1997). Upon conversion to its GTP-bound form during metaphase, its effector kinase Cdc7 (Sohrmann et al, 1998) is recruited, followed by Sid1–Cdc14 (Guertin et al, 2000). In anaphase B, the downstream SIN kinase, Sid2–Mob1, which localizes constitutively to SPBs, concentrates at the CR before constriction and is thought to transduce the signal to constrict (Sparks et al, 1999; Hou et al, 2000; Salimova et al, 2000). To prevent premature septation in interphase, a bipartite GAP complex comprising Cdc16 and Byr4 binds and inhibits Spg1 at SPBs (Song et al, 1996; Furge et al, 1998; Jwa and Song, 1998; Krapp et al, 2008). The GAP complex also regulates asymmetric distribution of SIN activity at the two SPBs throughout mitosis (Cerutti and Simanis, 1999; Li et al, 2000). In metaphase, Byr4–Cdc16 are absent from both SPBs, but are then recruited to the old SPB during anaphase B, thus permitting SIN activity only on the new SPB (Sohrmann et al, 1998; Grallert et al, 2004). Asymmetric distribution of SIN activity is critically important for the SIN to trigger septation precisely and also to silence the SIN after the completion of cytokinesis (Garcia-Cortes and McCollum, 2009). Two scaffold proteins, Sid4 and Cdc11, provide the spatial cues for assembly of the SIN and its regulators at SPBs (Krapp et al, 2001, 2003, 2004a; Tomlin et al, 2002; Morrell et al, 2004), while the conserved Polo-like kinase, Plo1, temporally regulates SIN signalling (Mulvihill et al, 1999; Tanaka et al, 2001). Among its other activities, Plo1 has a key role in forming the contractile ring, predicting the site of division, driving septum formation (Ohkura et al, 1995; Bahler et al, 1998), and plo1+ overexpression activates the SIN pathway (Ohkura et al, 1995; Mulvihill et al, 1999). To execute these events faithfully, Plo1 localization within the cell is controlled precisely. Plo1 concentrates on the mitotic, but not the interphase SPB (Mulvihill et al, 1999), partly through association with the SIN scaffold, Sid4 (Morrell et al, 2004), suggesting that Plo1 might directly target one or more SIN components to drive septum formation. A pathway homologous to the SIN, called the mitotic exit network (MEN), exists in Saccharomyces cerevisiae (for reviews see Bardin and Amon, 2001; McCollum and Gould, 2001; de Bettignies and Johnston, 2003; Seshan and Amon, 2004). While the Plo1 target at the SIN has not yet been identified in S. pombe, the S. cerevisiae Plo1 homologue, Cdc5, phosphorylates and inhibits the GAP complex, Bub2–Bfa1, allowing MEN activation (Hu et al, 2001; Geymonat et al, 2003). In addition to the SIN/MEN pathways that function during every cell cycle, multiple checkpoint pathways also control mitotic progression. For instance, in the event that chromosomes are not properly attached to the mitotic spindle during metaphase, the spindle assembly checkpoint (SAC) inhibits the anaphase-promoting complex/cyclosome to prevent anaphase onset and mitotic exit (for reviews see Malmanche et al, 2006; Varetti and Musacchio, 2008; Zich and Hardwick, 2010). In addition to the SAC, studies in yeast have identified a SAC-independent pathway required to inhibit cytokinesis when chromosomes are not properly attached to the mitotic spindle (Alexandru et al, 1999; Beltraminelli et al, 1999; Gardner and Burke, 2000). In S. pombe, one effector of the SAC-independent pathway is the checkpoint protein, Dma1 (Murone and Simanis, 1996; Guertin et al, 2002). Dma1 localizes to SPBs and the division site, and dma1+ overexpression prevents the SIN kinases from assembling at SPBs (Guertin et al, 2002). Furthermore, Dma1 binds the SIN scaffold, Sid4, and delays recruitment of Plo1 to SPBs during a checkpoint response (Guertin et al, 2002). However, the mechanism by which this occurs is unknown. At its N terminus, Dma1 has a Forkhead-associated (FHA) domain, which is predicted to interact with phosphothreonine residues (Durocher et al, 2000; Durocher and Jackson, 2002; Mahajan et al, 2008). At its C terminus, Dma1 contains a ring-finger (RF) domain, which most likely confers E3 ubiquitin ligase activity to the protein (for reviews see Joazeiro and Weissman, 2000; Deshaies and Joazeiro, 2009). The FHA domain is required for Dma1 localization to SPBs and the cell division site, and both domains are required for proper checkpoint function (Guertin et al, 2002). Dma1 belongs to a small class of proteins that encode both FHA and RF domains, of which there are two in humans, CHFR (Scolnick and Halazonetis, 2000) and RNF8 (Kolas et al, 2007). CHFR (CHeckpoint protein with FHA and RF domains) is a tumour suppressor protein, which has been implicated in a mitotic checkpoint termed the antephase checkpoint (Scolnick and Halazonetis, 2000; Matsusaka and Pines, 2004). RNF8 is also a checkpoint protein, which has a role in the DNA damage response pathway (Huen et al, 2007; Kolas et al, 2007; Wang and Elledge, 2007). Two homologues exist in S. cerevisiae, Dma1 and Dma2, which are functionally redundant and are required for the spindle position checkpoint (Fraschini et al, 2004). While all of these proteins share little sequence similarity outside of their FHA and RF domains, all participate in cell cycle checkpoints (for review see Brooks et al, 2008), which might imply a conserved mode of action. To elucidate the mechanism of Dma1 inhibition of the SIN, we screened all SIN components as potential Dma1 substrates and found that the SIN scaffold, Sid4, is ubiquitinated in a Dma1-dependent manner and that Sid4 ubiquitination is required to prevent cytokinesis during a mitotic checkpoint arrest. Furthermore, when the spindle checkpoint is activated in the absence of Sid4 ubiquitination, Plo1 prematurely accumulates at SPBs and the GAP component, Byr4, is driven off SPBs earlier compared with wild type cells. Our data indicate that Dma1 ubiquitinates the SIN scaffold protein, Sid4, to antagonize Plo1 localization and access to SIN substrates in order to delay cytokinesis. Results The SIN scaffold, Sid4, is ubiquitinated in vivo dma1+ encodes a RF domain, which is predicted to have E3 ubiquitin ligase activity (Figure 1A). To determine whether Dma1 is in fact a functional E3 ubiquitin ligase, Dma1 was tagged at its endogenous C terminus with HA3–TAP and purified from S. pombe lysates. When the TAP eluate was incubated with an E1-activating enzyme and the E2-conjugating enzyme, Ubc13–Uev1a, Dma1 catalysed formation of polyubiquitin chains in vitro (Figure 1B, left panel). To be sure that the polyubiquitin chains were formed in a Dma1-specific manner and were not a product of another E3 contaminant present in the TAP eluate, a conserved hydrophobic residue within the RF domain (I194) that is expected to disrupt interaction with its cognate E2 enzyme (Katoh et al, 2003) was mutated to alanine (Figure 1A). When the Dma1(I194A)–HA3–TAP eluate was incubated with the E1 and E2 enzymes, polyubiquitin chains were not formed (Figure 1B, right panel). Taken together, these data indicate that the predicted RF domain of Dma1 confers ubiquitin ligase activity to the protein. Figure 1.The SIN scaffold, Sid4, is ubiquitinated in vivo. (A) Schematic diagram of Dma1 protein with relative positions of Dma1 FHA and RF domains and the I194A point mutation indicated. (B) In vitro ubiquitination assay using an E1-activating enzyme, the human E2-conjugating enzyme, Ubc13/Uev1a and either dma1–HA3–TAP or dma1(I194A)–HA3–TAP purified from S. pombe lysates arrested by the nda3-KM311 mutation. (C) List of SIN and SPB proteins screened for in vivo ubiquitination. (D) In vivo ubiquitination assay of proteins listed in C. Each protein was purified from checkpoint-activated cells (nda3-KM311) and visualized by immunoblot using fluorescently labelled streptavidin (bottom panels) and a Ubiquitin antibody (top panels). Download figure Download PowerPoint Given that the Dma1 RF domain is required to maintain a spindle checkpoint arrest and that dma1+ antagonizes SIN signalling by perturbing Plo1 SPB localization (Guertin et al, 2002), we reasoned that Dma1 performed its checkpoint function by targeting Plo1 or other SIN component(s) for ubiquitination. Therefore, the in vivo ubiquitination status of Plo1 and every SIN component (Figure 1C) was examined in checkpoint-activated cells (Figure 1D). We also tested the ubiquitination status of the SPB component Ppc89, which is required for Sid4 association with the SPB, and Cut12, with which Plo1 also interacts at the SPB (Flory et al, 2002; MacIver et al, 2003) (Figure 1C). Each protein was tagged at its endogenous C terminus with a His6–BIO–His6 (HBH) epitope and purified from denatured lysates using Ni2+–NTA and streptavidin resin (Tagwerker et al, 2006). Proteins were purified from cells in which the spindle checkpoint had been activated using a reversible cold-sensitive mutation in the β-tubulin gene (nda3-KM311) (Hiraoka et al, 1984) and the ubiquitination status was determined by immunoblotting for ubiquitin. To validate that each protein was indeed purified, we also blotted with streptavidin, which recognizes the biotinylated epitope. Through this approach, we found that the SIN scaffold, Sid4, was the only protein tested to be robustly ubiquitinated in vivo (Figure 1D). Sid4 is ubiquitinated in a Dma1-dependent manner The finding that Sid4 is ubiquitinated in vivo during a checkpoint arrest suggests that it might be a Dma1 substrate. In this regard, it is noteworthy that Dma1 and Sid4 were shown previously to interact with each other by yeast two-hybrid analysis (Guertin et al, 2002). We therefore examined whether Sid4 ubiquitination required Dma1. Mutants were generated in which either the entire coding region of dma1+ was deleted or single mutations within the dma1+ coding region (R64 or I194) were mutated to alanine and integrated at the endogenous dma1+ locus (Figure 2A). Mutating R64 to alanine is predicted to disrupt interaction with phosphothreonine residues (Durocher and Jackson, 2002) and impedes localization of Dma1 to SPBs and the cell division site (Figure 2B, compare panels I and II), while the I194A mutation eliminates Dma1 E3 ligase activity (Figure 1B), but does not disrupt its localization to SPBs or the division site (Figure 2B, compare panels I and III). Figure 2.Sid4 ubiquitination requires Dma1 function. (A) Schematic diagram of Dma1 domains and positions of the R64A and I194A mutations. The R64A mutation prevents interaction with phosphothreonine motifs and I196A inactivates ubiquitin ligase activity. (B) Localization of dma1–GFP (panel I), dma1(R64A)–GFP (panel II) and dma1(I194A)–GFP (panel III) in cells growing in log phase. Scale bar, 5 μm. (C) Spindle checkpoint assay. Cells of the indicated strains were synchronized at 32°C in G2 by centrifugal elutriation, shifted to 18°C, and the septation index of each strain determined every 30 min for 9 h. (D) In vivo ubiquitination status of Sid4–HBH in nda3-KM311 dma1Δ or nda3-KM311 dma1 mutants. Download figure Download PowerPoint To validate that the dma1 mutants compromise Dma1 function, each dma1 mutant was combined with the nda3-KM311 mutation and tested for checkpoint function. Cells were synchronized in G2 by centrifugal elutriation, shifted to the restrictive temperature (18°C) to activate the spindle checkpoint, and septation indices were measured at 30 min intervals for 9 h. While the nda3-KM311 dma1+ strain maintained a checkpoint arrest for ∼7 h, the nda3-KM311 dma1(R64A) and nda3-KM311 dma1(I194A) mutant strains could not maintain an arrest and formed aberrant septa at ∼5 h, which is comparable with nda3-KM311 dma1Δ cells (Figure 2C). Thus, the R64A and I194A mutations compromise Dma1-dependent checkpoint function. We next examined Sid4 ubiquitination in checkpoint activated (nda3-KM311) dma1Δ, dma1(R64A) and dma1(I194A) mutants. Cells were shifted to 18°C for 5 h to activate the spindle checkpoint and Sid4 ubiquitination was examined. Strikingly, in the absence of Dma1 protein, activity or localization, Sid4 ubiquitination was abolished (Figure 2D). These data indicate that Sid4 is ubiquitinated in a Dma1-dependent manner. Sid4 ubiquitination is required for Dma1-dependent checkpoint function To determine if Sid4 ubiquitination is required for the Dma1-dependent checkpoint arrest, a ubiquitination deficient sid4 allele was generated. Ubiquitin transfer often occurs in a sequence-independent manner and can occur on multiple substrate lysines, making site identification challenging (for reviews see Laney and Hochstrasser, 1999; Pickart, 2001). Sid4 contains 49 lysines (Figure 3A, top diagram) and mutating all 49 sites simultaneously would likely disrupt protein function. Thus, four sid4 mutants were made, in which clusters of lysine residues were mutated that, collectively, cover every lysine within Sid4 (Supplementary Figure 1A). As sid4+ is essential for viability, we first tested whether the four mutants could rescue the temperature-sensitive sid4–SA1 mutant at the restrictive temperature (data not shown) and as they all could, each was then integrated at the endogenous sid4+ locus to examine its in vivo ubiquitination status. Surprisingly, all four mutants were still ubiquitinated in vivo (Supplementary Figure 1B). Therefore, in order to create a ubiquitin-deficient sid4 allele, we needed to generate a mutant that would eliminate more lysine residues simultaneously. However, all four mutants generated above were severely cold sensitive (data not shown), indicating that Sid4 function was already compromised and adding more mutations would likely exacerbate these phenotypes. Figure 3.Sid4 ubiquitination is required to maintain a checkpoint arrest. (A) Schematic diagrams of Sid4 with relative positions of all 49 lysines (top), Ppc89 (middle) and the Sid4N–Ppc89C fusion mutant (bottom). Predicted coiled-coil regions are shown in black. (B) In vivo ubiquitination of Sid4–HBH and Sid4N–Ppc89C–HBH. (C) Localization of Sid4N–Ppc89C–GFP (panel I), Cdc11–GFP (panel II) and Dma1–GFP (panel III) in sid4N–ppc89C–HBH mutant cells. Scale bar, 5 μm. (D) Spindle checkpoint assay. Cells of the indicated strains were blocked at 32°C in S phase with hydroxyurea, released into hydroxyurea-free media at 18°C, and the septation index of each strain was determined every 30 min for 9 h. (E) Cells from each of the strains examined in 3D at the 7 h time point stained with methyl blue, which stains the septa, and DAPI, which stains DNA. (^) indicate septated cells that have bypassed the checkpoint. Scale bar, 5 μm. Download figure Download PowerPoint Thus, as an alternative means of eliminating relevant Sid4 lysine residues without disrupting protein function, we made use of previous structure and function analyses of Sid4 and the core SPB protein, Ppc89. The N-terminal 300 amino acids of Sid4 are required for direct binding to Plo1 (Morrell et al, 2004), Cdc11 (Tomlin et al, 2002) and Dma1 (Guertin et al, 2002), indicating that this region contains the essential SIN scaffolding activity of Sid4. The C termini of both Sid4 and Ppc89 contain several predicted coiled-coil regions (Figure 3A, top and middle diagram, respectively), which are only required for their SPB localization (Rosenberg et al, 2006). In fact, replacing the Sid4 C terminus with the SPB targeting region of Ppc89 (Figure 3A, bottom diagram) rescues both the temperature-sensitive sid4–SA1 allele at 36°C and the sid4Δ (Rosenberg et al, 2006). Thus, the sid4N–ppc89C fusion mutant, which eliminates ∼76% of Sid4 lysines on the protein, was integrated at the endogenous sid4+ locus and tested for in vivo ubiquitination. While Sid4–HBH was robustly ubiquitinated, ubiquitination of the Sid4N–Ppc89C–HBH mutant was essentially eliminated (Figure 3B). Importantly, sid4N–ppc89C mutant cells were wild type for morphology and were not temperature sensitive. These data indicate that Dma1 targets the Sid4 C terminus for ubiquitination in vivo. As expected, Sid4N–Ppc89C–GFP localized to SPBs properly (Figure 3C, panel I) and Cdc11–GFP, whose localization depends on Sid4, also localized to SPBs normally (Figure 3C, panel II). Furthermore, Cdc11–GFP intensities at SPBs are not significantly altered in sid4N–ppc89C mutant cells compared with wild-type cells (Supplementary Figure 2A and B). Thus, as predicted by its wild type morphology, the Sid4N–Ppc89C mutant does not disrupt the SIN scaffold complex. To ensure that the loss of Sid4N–Ppc89C ubiquitination was not due to a failure to recruit Dma1, we examined Dma1–GFP localization and found that it was present at SPBs in sid4N–ppc89C mutant cells (Figure 3C, panel III), consistent with the previous observation that Dma1 interacts with the Sid4 N-terminal 300 amino acids (Guertin et al, 2002). Thus, while the Sid4 N terminus binds Dma1, its C terminus is required for ubiquitination. Collectively, these data indicate that the sid4N–ppc89C mutant retains full scaffolding and essential SIN functions of sid4+, but is unable to be ubiquitinated in vivo even in the presence of Dma1. We then assessed the checkpoint function of the sid4N–ppc89C mutant. Cells were arrested in S phase with hydroxyurea (HU), released synchronously at 18°C to activate the spindle checkpoint, and septation indices were measured at 30 min intervals for 9 h. The nda3-KM311 strain maintained a checkpoint arrest for ∼7 h (Figure 3D and E, top left panel); however, the nda3-KM311 sid4N–ppc89C mutant formed aberrant septa (marked with (^) in Figure 3E, bottom left panel) at ∼5 h, which phenocopied the dma1–RF mutant (nda3-KM311 dma1(I194A)) (Figure 3D and E, top right panel). Importantly, a double dma1(I194A) sid4N–ppc89C mutant septated with similar kinetics as either mutant alone and did not display any other additive effects (Figure 3D and E, bottom right panel), suggesting that these mutants bypass a checkpoint arrest via the same mechanism. Thus, Sid4 ubiquitination is necessary to inhibit cytokinesis during a dma1-dependent checkpoint arrest. Sid4 ubiquitination antagonizes Plo1 recruitment to SPBs during a checkpoint response When the spindle checkpoint is activated in the absence of dma1+, Plo1 is recruited to SPBs earlier (Guertin et al, 2002). Because our data suggest that Dma1 ubiquitinates Sid4 when a mitotic checkpoint is activated, we tested if Sid4 ubiquitination was the biochemical signal that perturbs Plo1 recruitment to SPBs by measuring the timing of Plo1 recruitment to SPBs in checkpoint-activated sid4N–ppc89C cells. Endogenously expressed Plo1 fused to a single GFP is difficult to visualize in vivo. Thus, to improve visualization three tandem copies of GFP were fused to the C terminus of Plo1 (Plo1–GFP3) and used in the subsequent experiments. nda3-KM311, nda3-KM311 dma1(I194A) and nda3-KM311 sid4N–ppc89C cells were synchronized in G2 by lactose gradient sedimentation, shifted to 18°C to activate the spindle checkpoint, and Plo1–GFP3 was visualized at 30 min intervals for 9 h. In dma1+ cells, Plo1–GFP3 was not visible on SPBs until ∼4 to 5 h (Figure 4A and D). However, in the dma1(I194A) mutant, Plo1–GFP3 was detected at SPBs ∼2 h earlier compared with dma1+ cells and cells failed to arrest in mitosis (Figure 4B and D), which is similar to the premature recruitment observed previously for dma1Δ cells (Guertin et al, 2002). Similarly, Plo1–GFP3 was recruited to SPBs earlier in sid4N–ppc89C mutant cells (Figure 4C and D). It should be noted that when cells are arrested in prometaphase by the nda3-KM311 mutation, Plo1 localizes to both SPBs; however, because the mitotic spindle does not form and SPBs do not separate in this arrest, Plo1's signal in the later time points is slightly obscured by the fact that it is localizing on two SPBs that are sometimes overlapping in the Z axis. To be sure that we were quantitating SPB-localized Plo1, Plo1–GFP3 was colocalized with the constitutive SPB marker, Sad1–mCherry (Hagan and Yanagida, 1995; Figure 4A–C, right panels). These data suggest that when the spindle checkpoint is activated, Sid4 ubiquitination antagonizes Plo1 recruitment to SPBs and thereby prevents it from reaching its substrates and activating the SIN. Figure 4.Sid4 ubiquitination delays Plo1 recruitment to the SPBs when the spindle checkpoint is activated. (A–C) nda3-KM311 (A), nda3-KM311 dma1(I194A) (B) or nda3-KM311 sid4N–ppc89C (C) cells were synchronized at 32°C in G2 by lactose gradient sedimentation, released to 18°C to activate the spindle checkpoint, and Plo1-GFP3 and Sad1-mCherry localization at the SPBs were imaged periodically for 9 h. In each panel, the images on the left show Plo1–GFP3 localization alone and the images on the right show merged images of Plo1–GFP3 colocalized with Sad1–mCherry at each of the times indicated. Scale bar, 10 μm. (D) The kinetics of Plo1 recruitment to SPBs was measured for each of the strains shown by calculating the percentage of cells with Plo1–GFP3 on SPBs at each time point. Download figure Download PowerPoint Sid4 ubiquitination antagonizes Plo1 recruitment to SPBs during interphase As Dma1 can be detected at SPBs in the absence of checkpoint induction, we examined a potential role for Sid4 ubiquitination during normal cell cycle progression. While Sid4 was most robustly ubiquitinated during a mitotic arrest, as expected, it was also ubiquitinated in G2 cells, but significantly less ubiquitination was detected during S phase (Figure 5A). As Sid4 ubiquitination levels fluctuate throughout the cell cycle, we tested if Dma1 concentration at SPBs was also cell cycle dependent by measuring Dma1–GFP intensities at SPBs in different cell cycle stages and comparing these intensities with the constitutive SPB marker Sid4–RFP (Morrell et al, 2004). Dma1–GFP intensity was detected at low levels in prometaphase cells grown to log phase under permissible conditions (Figure 5B and C) and was significantly increased during a mitotic checkpoint arrest (nda3-KM311 arrest) (Figure 5B and C), suggesting that Dma1 concentrates at SPBs in response to mitotic stress. Dma1-GFP was also detected in cells arrested in G2 (cdc25-22) (Figures 5B and C), although with significantly decreased intensity compared with nda3-KM311-arrested cells, and it was not detected on SPBs in cells arrested in S phase (Figure 5B and C). Thus, the levels of Sid4 ubiquitination correlate with the concentration of SPB-localized Dma1. Figure 5.Sid4 ubiquitination prevents Plo1 recruitment to SPBs during interphase. (A) In vivo ubiquitination of Sid4–HBH in asynchronous cells or cells arrested in G2 (cdc25-22), prometaphase (nda3-KM311) or S phase (hydroxyurea; HU). (B) Representative images showing Dma1–GFP and Sid4–RFP localization in a G2 arrest (cdc25-22 arrest), an S-phase arrest (HU arrest), prometaphase cell growing in log phase, and a mitotic arrest when the checkpoint is active (nda3-KM311 arrest). Scale bar, 5 μm. (C) Quantitation of relative Dma1–GFP/Sid4-RFP intensity ratios for each of the cell cycle stages shown in B plotted as arbitrary units. For each cell cycle stage, Dma1–GFP and Sid4–RFP intensities were measured for at least 20 cells and averaged; error bars represent standard error of the mean, *P<0.05. (D) Representative images showing Plo1–GFP3 and Sad1–mCherry localization at SPBs during a cdc25-22 arrest in wild type (left panels), dma1Δ (middle panels) and sid4N–ppc89C (right panels) cells. Scale bar, 5 μm. (E) Quantitation of relative Plo1–GFP3/Sad1–mCherry intensity ratios at SPBs for each of the strains shown in D plotted in arbitrary units. For each strain, Plo1–GFP3 and Sad1–mCherry intensities were measured for at least 20 cells and averaged; error bars represent standard error of the mean, *P<0.05. Download figure Download PowerPoint Plo1 localization to SPBs is also cell cycle regulated, accumulating at SPBs upon commitment to mitosis (Mulvihill et al, 1999). As Sid4 ubiquitination antagonizes Plo1 localization at SPBs and Sid4 is ubiquitinated in interphase cells, we wondered if the absence of Sid4 ubiquitination would allow Plo1 to concentrate at SPBs in interphase. Thus, dma1+, dma1Δ or sid4N–ppc89C cells were arrested in G2 using the temperature-sensitive cdc25-22 mutation, and Plo1–GFP3 intensities at SPBs were measured relative to Sad1–mCherry. While Plo1–GFP3 was only detected at low levels in dma1+ cells (Figure 5D and E), Plo1–GFP3 intensities at SPBs were significantly increased in dma1Δ cells (Figure 5D and E). A similar increase in Plo1–GFP3 intensities was observed in sid4N–ppc89C cells, in which Sid4 ubiquitination is abolished (Figure 5D and E). These data suggest that Sid4 ubiquitination antagonizes Plo1 localization to SPBs during interphase and during a mitotic checkpoint arrest. Byr4 is a potential Plo1 target While the direct SIN target(s) of Plo1 have not yet been identified in S. pombe, the S. cerevisiae Plo1 homologue, Cdc5, is known to phosphorylate and inhibit the Byr4 ortholog and GAP component Bfa1, resulting in MEN activation (Hu et al, 2001; Geymonat et al, 2003). Bfa1 phosphorylation by Cdc5 inhibits its GAP activity in vitro and also ejects it from SPBs (Hu et al, 2001; Geymonat et al, 2003). S. pombe Byr4 is also a phosphoprotein (Song et al, 1996) and is hyperphosphorylated just before septation (Krapp et al, 2008). Thus, the potential of Plo1 SPB recruitment influencing Byr4 phosphorylation status and SPB localization was examined. First, Byr4 was tested as a Plo1 substrate in vitro. MBP and MBP–Byr4 were produced in E. coli, and purified on amylose resin. When purified proteins were incubated with Plo1 purified from baculovirus-infect