Tem1 localization to the spindle pole bodies is essential for mitotic exit and impairs spindle checkpoint function

JCB: Article The Rockefeller University Press $30.00 J. Cell Biol. Vol. 192 No. 4 599–614 www.jcb.org/cgi/doi/10.1083/jcb.201007044 JCB 599 Correspondence to Fernando Monje-Casas: [email protected] Abbreviations used in this paper: DIC, differential interference contrast; FEAR, Cdc14 early anaphase release; GAP, GTPase-activating protein; MEN, mitotic exit network; SAC, spindle assembly checkpoint; SPB, spindle pole body; SPOC, spindle position checkpoint. Introduction After the genome is duplicated and chromosomes are distributed between the mother and daughter cells during anaphase, cells prepare to exit from mitosis. Mitotic exit is determined by the inactivation of mitotic Cdks (for review see Stegmeier and Amon, 2004). In Saccharomyces cerevisiae, Cdk inactivation is triggered by the phosphatase Cdc14 (Visintin et al., 1998). Cdc14 is sequestered in the nucleolus from G1 to metaphase, and only at anaphase onset is it released from its inhibitor, Cfi1/ Net1 (Shou et al., 1999; Visintin et al., 1999). Once Cdc14 is released, it reverses phosphorylation events promoted by mitotic Cdks, which eventually determines their inactivation. Two different signaling pathways control the release of Cdc14 from the nucleolus. The Cdc14 early anaphase release (FEAR) network promotes an initial release of the phosphatase in the first stages of anaphase (Rock and Amon, 2009). However, a second signaling cascade, the mitotic exit network (MEN), is necessary to keep a sustained release of Cdc14 and to fully inactivate mitotic Cdk activity (Jaspersen et al., 1998; Lee et al., 2001; Stegmeier and Amon, 2004). Tem1 is a GTPase that initiates MEN signaling (Shirayama et al., 1994; Lee et al., 2001). The two-component GTPaseactivating protein (GAP) Bfa1-Bub2 is a negative regulator of Tem1 (Pereira et al., 2000; Geymonat et al., 2002). Tem1, Bfa1, and Bub2 localize to the spindle pole bodies (SPBs), the equivalent of the centrosomes in yeast (Pereira et al., 2000). Nud1, a component of the SPB, functions as an anchor for MEN components to this structure (Gruneberg et al., 2000). Bfa1 and Bub2 are thought to keep Tem1 in an inactive GDP-bound state during most of the cell cycle (Bardin et al., 2000; Pereira et al., 2000; Geymonat et al., 2002). The kinase Kin4 maintains the GAP in an active state by preventing the inhibitory phosphorylation of Bfa1 by the polo-like kinase Cdc5 (Hu et al., 2001; D’Aquino et al., 2005; Pereira and Schiebel, 2005; Maekawa et al., 2007). At anaphase onset, one of the SPBs enters the daughter cell, and inhibition of Tem1 by Bfa1-Bub2 is alleviated. At the same time, MEN signaling is stimulated by Lte1, a protein that localizes to the bud cortex (Bardin et al., 2000; Pereira et al., 2000). It is not yet known how this protein activates the MEN, but its role in mitotic exit is not mediated by a modification of Tem1 activity (Geymonat et al., 2009). Once Tem1 is activated, it initiates a signaling cascade that includes the Cdc15 and Dbf2 kinases (Jaspersen et al., 1998; Lee et al., 2001). Dbf2, together with its associated factor Mob1, promotes Cdc14 release from The mitotic exit network (MEN) is a signaling cascade that triggers inactivation of the mitotic cyclindependent kinases and exit from mitosis. The GTPase Tem1 localizes on the spindle pole bodies (SPBs) and initiates MEN signaling. Tem1 activity is inhibited until anaphase by Bfa1-Bub2. These proteins are also part of the spindle position checkpoint (SPOC), a surveillance mechanism that restrains mitotic exit until the spindle is correctly positioned. Here, we show that regulation of Tem1 localization is essential for the proper function of the MEN and the SPOC. We demonstrate that the dynamics of Tem1 loading onto SPBs determine the recruitment of other MEN components to this structure, and reevaluate the interdependence in the localization of Tem1, Bfa1, and Bub2. We also find that removal of Tem1 from the SPBs is critical for the SPOC to impede cell cycle progression. Finally, we demonstrate for the first time that localization of Tem1 to the SPBs is a requirement for mitotic exit. Tem1 localization to the spindle pole bodies is essential for mitotic exit and impairs spindle checkpoint function Mauricio Valerio-Santiago1,2 and Fernando Monje-Casas1,2 1Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER) and 2Departamento de Genética, Universidad de Sevilla, E-41092 Sevilla, Spain © 2011 Valerio-Santiago and Monje-Casas This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/). THE JOURNAL OF CELL BIOLOGY JCB • VOLUME 192 • NUMBER 4 • 2011 600 MEN signaling, we constitutively targeted Tem1 to SPBs. Cnm67 is an integral component of the outer plaque of the SPB (Schaerer et al., 2001). A fusion of Cnm67 and Tem1 forced the GTPase to constitutively load onto SPBs. Localization of this chimera was found to be symmetric from SPB duplication until the end of mitosis (Fig. 1, A and B). Localization of Bfa1 is similar to that of Tem1, but Bfa1 is more stable on the dSPB and more asymmetric than Tem1 in late anaphase (Molk et al., 2004; Monje-Casas and Amon, 2009). To achieve a constitutive targeting of Tem1 to the SPBs but in an asymmetric manner, we also fused Tem1 and Bfa1. Localization of the Bfa1–Tem1 chimera resembled that of Bfa1: the protein started to localize asymmetrically in metaphase (Fig. 1 A), and this asymmetry was completely established by anaphase (Fig. 1 B). Cells carrying TEM1 fused to a degron module, and under the control of the GAL1-10 promoter (GAL-UPL-TEM1; Shou et al., 1999), as the only source for Tem1, were transformed with centromeric plasmids expressing TEM1, CNM67–TEM1, or BFA1–TEM1 under the control of the endogenous TEM1 promoter. The levels of expression for each protein are shown in Fig. S1 A. The growth of GAL-UPL-TEM1 cells in glucosecontaining media was similar for cells carrying the plasmids with the Tem1 chimeras or a plasmid with the wild-type copy of the TEM1 gene (Fig. 1 C). Our results show that constitutive targeting of Tem1 to the SPBs does not have a great impact on the viability of cells, regardless of whether the protein localizes in a symmetric or a mainly asymmetric manner. Cnm67–Tem1 and Bfa1–Tem1 chimeras increase the residence time of Tem1 on SPBs To determine the dynamicity of the Tem1 chimeras on the SPBs, we performed FRAP experiments. The half-recovery time for N-terminally eGFP-tagged Tem1 (eGFP-Tem1) on SPBs in metaphase cells was 3.4 s, and 62% of the total signal was recovered after photobleaching (Fig. 2 A). No difference was observed in the dynamics of eGFP-Tem1 loading onto the SPBs between metaphase and anaphase cells (Fig. 2, A and B). These results are highly similar to those obtained previously for the C-terminally tagged Tem1-eGFP (half-recovery time = 4 s; percentage recovery = 60–80%; Monje-Casas and Amon, 2009). When Tem1 was fused to Cnm67, the dynamics of loading onto the SPBs changed dramatically. No recovery of the eGFP signal could be detected for the fusion during the extension of the FRAP experiment (2 min; Fig. 2 C). The increase in the residence time of the protein on the SPB indicated that the turnover of Cnm67–Tem1 in this structure was extremely reduced. This reduced exchange rate of Tem1 on the SPBs as a consequence of its fusion to Cnm67 was observed both in metaphase and in anaphase (Fig. 2, C and D). The mobility of Bfa1 on the SPBs differs from that of Tem1 (Caydasi and Pereira, 2009; Monje-Casas and Amon, 2009). Bfa1-eGFP is largely immobile on the dSPB. Likewise, the Bfa1–Tem1 fusion showed an increased residence time on the dSPB in comparison to Tem1 (Fig. 2, E and F). Again, no differences in the dynamics of protein loading onto the SPBs the nucleolus and maintains the phosphatase in the cytoplasm (Mohl et al., 2009). Bfa1-Bub2 localizes asymmetrically to the SPB that enters the daughter cell during anaphase (dSPB; Bardin et al., 2000; Pereira et al., 2000). Tem1 localization to the SPBs is highly dynamic (Molk et al., 2004; Monje-Casas and Amon, 2009), and it has been shown to be dependent on both Bfa1 and Bub2 (Pereira et al., 2000). Tem1 is also enriched on the dSPB during anaphase, but it is not as asymmetric as Bfa1-Bub2 (Bardin et al., 2000; Molk et al., 2004). Based on its localization, it has been assumed that Tem1 signals mitotic exit from the SPB. However, this has not yet been formally demonstrated. To properly distribute the genetic material, the spindle must be correctly positioned along the mother–daughter cell axis. The spindle position checkpoint (SPOC) is a surveillance mechanism that verifies the location of the mitotic spindle and restrains cell cycle progression until one SPB enters the daughter cell in anaphase (for review see Lew and Burke, 2003). Bfa1, Bub2, and Kin4 are key members of the SPOC. Upon SPOC activation, Kin4 loads onto both SPBs and increases the turnover of Bfa1-Bub2 on this structure (Caydasi and Pereira, 2009). A stable association of the GAP with the SPBs interferes with the function of the SPOC (Caydasi and Pereira, 2009). A redistribution model has been proposed by which malfunction of the SPOC could be based on a reduction of the cytoplasmic pool of Bfa1 and Bub2 that would normally spread throughout the cytoplasm, inhibiting mitotic exit (Caydasi and Pereira, 2009). Despite numerous studies to analyze the effects of interfering with the normal localization of Bfa1-Bub2 to the SPBs, little is known about the consequences of altering the dynamics and the pattern of Tem1 localization on cell cycle progression and the SPOC function. Here, we investigate the effects of differential targeting of the GTPase and provide new insights on the regulation of the MEN and the SPOC. We find that an increase in the residence time of Tem1 on the SPBs determines the recruitment of other MEN components to this structure, whereas it does not lead to a precocious mitotic exit due to the concerted action of Bfa1 and Clb2. Additionally, we reevaluate the interdependence of Bfa1, Bub2, and Tem1 localization, and show that Tem1 can still load onto SPBs in the absence of the GAP and that Tem1 can alter the pattern of Bfa1 localization. By constitutive targeting of Tem1 to the SPBs, we also demonstrate that removal of this protein from the spindle poles is critical for SPOC function. Thus, we propose an exclusion model by which the increase observed in the turnover of Bfa1-Bub2 on the SPBs after SPOC activation contributes to impede the association of Tem1 to this structure. Finally, we demonstrate for the first time that localization of Tem1 to the SPBs is a requirement for mitotic exit. Results Constitutive targeting of Tem1 to SPBs Tem1 is a highly mobile protein that localizes preferentially to the SPB that enters the daughter cell during anaphase (dSPB; Molk et al., 2004; Monje-Casas and Amon, 2009). To determine the role of Tem1 localization and dynamicity on the SPBs in 601Tem1 dynamics on spindle pole bodies regulate mitotic exit • Valerio-Santiago and Monje-Casas both Cnm67–Tem1 and Bfa1–Tem1, the amounts of Tem1 on the SPBs increased compared with wild-type cells (Fig. S1 B). Thus, our results show that constitutive targeting of Tem1 to the SPBs by its fusion to Cnm67 or Bfa1 increases the residence time of the protein on the SPB and reduces its turnover in this structure. could be observed between metaphase and anaphase (Fig. 2, E and F). As previously shown for Bfa1 (Monje-Casas and Amon, 2009), the turnover of Bfa1–Tem1 on the SPBs increased in kar9 cells with mispositioned spindles (half recovery time = 24.4 s; percentage recovery = 41.2; Fig. S1 C). In agreement with the increased residence time observed for Figure 1. Constitutive targeting of Tem1 to the SPBs. GAL-UPL-TEM1 cells expressing eGFP-TEM1 (F567), eGFP-BFA1–TEM1 (F577), or eGFP-CNM67– TEM1 (F575) from a CEN plasmid were grown on 2% raffinose/2% galactose. (A and B) Localization of Tem1 and the chimeras (eGFP, green) in metaphase (A) and in anaphase (B) after cells were transferred to medium with 2% glucose. Nuclear morphology was assessed by DAPI (blue). A differential interference contrast (DIC) image is also shown. Loading of the eGFP-tagged protein to only one, to both (either asymmetrically [1 strong /1 weak] or symmetrically), or to none of the SPBs was quantified for each strain. Error bars represent SD (n = 3). Bars, 5 µm. (C) Viability of the cells was determined by spotting 10-fold serial dilutions of the original culture on plates without uracil and with either 2% glucose or 2% galactose/2% raffinose. Plates were incubated at 25°C. Cells carrying an empty vector (F797) were used as a control. JCB • VOLUME 192 • NUMBER 4 • 2011 602 Figure 2. The Tem1 chimeras increase the residence time of Tem1 on SPBs. FRAP analysis in GAL-UPL-TEM1 cells expressing eGFP-TEM1 (A and B; F567), eGFP-CNM67–TEM1 (C and D; F575), or eGFP-BFA1–TEM1 (E and F; F577) and growing on 2% glucose. Images of representative experiments are shown before (Pre-bleach), immediately after (0 s), and 120 s after (120 s) the laser pulse. The cell shape is outlined in white and an arrow indicates the bleached SPB. Graphs show the percentage of initial eGFP signal recovered with time in metaphase (A, C, and E) and anaphase (B, D, and F). Error bars indicate SD (n = 10). The red line represents fitting of the data to an exponential function. Bar, 5 µm. 603Tem1 dynamics on spindle pole bodies regulate mitotic exit • Valerio-Santiago and Monje-Casas in these cells, although Cdc15 gets prematurely loaded onto SPBs. Viability of the cells expressing Cnm67–Tem1 was not affected even when the initial release of Cdc14 by the FEAR is promoted by inactivation of the protein phosphatase 2A, its regulatory subunit Cdc55, or the Swe1 kinase (Fig. S2, B and C; Queralt et al., 2006; Liang et al., 2009). The mechanisms that restrain untimely MEN activation in cells that constitutively load Tem1 on the SPBs could be acting either upstream of Tem1 or directly on or downstream of Cdc15. We first checked whether viability of the cells expressing Cnm67–Tem1 was dependent on the known MEN inhibitors. Deletion of either BFA1 or KIN4 in cells expressing Cnm67– Tem1 did not have any effect on their viability (Fig. 4 A). Cell cycle progression was not affected in the absence of Bfa1 (Fig. 4 B), and Cdc14 was also released on time (Fig. S2 D). Although Bfa1 was not required for the viability of Cnm67–Tem1 cells, we examined whether its localization was affected by constitutive loading of Tem1 onto both SPBs. We quantified the localization of Bfa1 both in wild-type cells and in cells expressing Cnm67– Tem1. Surprisingly, although Bfa1 was mainly asymmetrically localized in wild-type cells, localization of Bfa1 became mostly symmetric in Cnm67–Tem1 cells, both in metaphase (Fig. 4 C) and in anaphase (Fig. 4 D). Localization of Tem1 to SPBs has been shown to depend on both Bfa1 and Bub2 (Pereira et al., 2000). However, loading of Bfa1 and Bub2 still occurs in cells lacking Tem1 (Pereira et al., 2000). Our results demonstrate that, despite being dispensable for Bfa1 loading onto SPBs, Tem1 can influence Bfa1 localization. It has been recently proposed that Clb2-Cdk negatively regulates the binding of Cdc15 to the mother SPB (mSPB) and inhibits Mob1, the associated factor of Dbf2 (König et al., 2010). Therefore, we tested whether viability of the cells expressing Cnm67–Tem1 was dependent on Clb2-Cdk activity. Deletion of CLB2 had no effect on the viability of the cells when Tem1 was constitutively loaded on the SPBs (Fig. 4 E). However, when we also deleted BFA1 in these cells, their viability was extremely impaired (Fig. 4 E), and they showed defects in cell cycle progression consistent with premature MEN activation (unpublished data). It is worth noting that deletion of CLB2 and BFA1 in an otherwise wild-type background had no effect on the viability of the cells (Fig. 4 E). Thus, premature mitotic exit of clb2 bfa1 cells is only promoted if Tem1 accumulates on the SPBs. Our results show that the viability of cells with Tem1 constitutively loaded onto SPBs is dependent on the concerted action of Bfa1-Bub2 and Clb2-Cdk. Either one of these regulators of the MEN is sufficient to fully sustain the viability of cells expressing the Tem1 fusions, but the abrogation of both activities highly impairs their growth. Constitutive loading of Tem1 onto SPBs impairs the SPOC, but not the spindle assembly checkpoint (SAC) Bfa1 and Bub2 also play an important role in the SPOC. The SPOC prevents exit from mitosis until the anaphase nucleus is correctly positioned (for review see Lew and Burke, 2003). Although neither progression through the cell cycle nor viability of the cells were greatly affected, we examined whether Increased residence time of Tem1 on SPBs leads to premature Cdc15 loading but not to an early Cdc14 release Constitutive targeting of Tem1 to the SPBs, both in a symmetric (Cnm67–Tem1) and in a mainly asymmetric (Bfa1–Tem1) manner, greatly modifies the dynamics of Tem1 loading onto SPBs without having a major impact on the viability of the cells. To further determine the effects of the constitutive loading of Tem1 onto SPBs, we examined cell cycle progression in cells carrying the Tem1 fusions as the only source of Tem1. We constructed cells with the endogenous TEM1 gene deleted and the CNM67–TEM1 or BFA1–TEM1 fusions integrated into the genome. The integration of the gene fusions in a single copy was checked by Southern blotting (unpublished data). As previously shown for GAL-UPL1-TEM1 cells transformed with plasmids encoding the chimeras (Fig. 1 C), integration of CNM67–TEM1 or BFA1–TEM1 allowed for the growth of tem1 cells with no apparent loss of viability (Fig. S2 A). Wild-type and tem1 cells expressing Cnm67–Tem1 or Bfa1–Tem1 were synchronized in G1 using a pheromone. After removal of the pheromone, cells were allowed to enter the cell cycle in a synchronous manner. The cells carrying the Tem1 chimeras progressed through the cell cycle as wild-type cells, and no premature entry into anaphase or mitotic exit delay could be observed (Fig. 3 A). To further characterize these cells, we checked the localization of other MEN components. Previous data suggest that loading of Cdc15 onto the SPBs triggers activation of the MEN (Visintin and Amon, 2001). In wild-type cells carrying a 3HA-tagged version of Cdc15, no signal could be observed for this protein on the SPBs until cells entered anaphase (Fig. 3, B and C; Visintin and Amon, 2001). However, cells carrying either of the Tem1 fusions showed premature Cdc15 loading onto the SPBs during metaphase (Fig. 3, B and C). Localization of Cdc15 at the SPBs followed the expected symmetric or asymmetric pattern according to Tem1 localization. Because Cdc15 localized to SPBs in metaphase cells expressing either of the chimeras, its premature loading must be caused by the increased residence time of Tem1 on the SPBs observed for both. Our data indicate that an increased residence time of Tem1 on the SPBs leads to untimely Cdc15 loading, but not to a premature entry into anaphase. To solve this apparent discrepancy, we also checked Dbf2 and Cdc14 localization in cells carrying the Tem1 chimeras. In agreement with the dynamics of progression through the cell cycle, and in contrast to what we observed for Cdc15, no premature loading of Dbf2 was detected in Cnm67– Tem1 or Bfa1–Tem1 cells (unpublished data), and Cdc14 was released from the nucleolus with the same kinetics as in wildtype cells (Fig. 3 D). Thus, our results show that an increased residence time of Tem1 on the SPBs by its constitutive targeting to this structure leads to premature Cdc15 loading but not to an early Cdc14 release, and suggest that additional mechanisms must exist that restrain MEN activity until anaphase onset. Viability of the Tem1 chimeras is dependent on the concerted action of Bfa1 and Clb2 The timely release of Cdc14 in cells carrying the Tem1 fusions suggests that MEN activity must be inhibited during metaphase JCB • VOLUME 192 • NUMBER 4 • 2011 604 Figure 3. Increased residence time of Tem1 on SPBs leads to premature Cdc15 loading but not to an early Cdc14 release. (A) Wild-type (F663) and tem1 cells carrying eGFP-BFA1–TEM1 (F679) or eGFP-CNM67–TEM1 (F667) fusions integrated at the URA3 locus were grown in rich media with 2% glucose (yeast peptone dextrose [YPD]) at 30°C, arrested in G1 with pheromone, and released into fresh media. Cell cycle progression was determined by spindle (tubulin) and nuclear morphology (DAPI). Percentages of metaphase and anaphase cells are shown for each time point. (B and C) Percentage of Cdc15-3HA loading onto the dSPB, mSPB, both SPBs, or none (B) for wild-type (F94) and cells expressing Bfa1–Tem1 (F678) or Cnm67–Tem1 (F677), 605Tem1 dynamics on spindle pole bodies regulate mitotic exit • Valerio-Santiago and Monje-Casas as determined by immunofluorescence. Error bars indicate SD (n = 3). (C) Representative images showing Cdc15-3HA (red), tubulin (green), and DAPI (blue) for metaphase cells are also presented. (D, left) Percentage of cells with 3HA-Cdc14 sequestered, partially released, or fully released for the same cells shown in A (key is shown on the bottom right). (D, right) Representative images showing 3HA-Cdc14 (red), tubulin (green), and DAPI (blue) are also presented. Bars, 5 µm. Figure 4. Viability of the chimeras depends on Bfa1 and Clb2. (A) Wild-type (F496) and tem1 cells expressing Cnm67–Tem1 alone (F637) or in combination with BFA1 (F657) or KIN4 (F745) deletions were grown on YPD at 25°C. Cells were plated on YPD in 10-fold serial dilutions spots and grown at 25°C. (B) Wild-type (F665), tem1 CNM67–TEM1 (F666), and tem1 CNM67–TEM1 bfa1 (F786) cells were arrested in G1 and released into fresh YPD. The percentages of metaphase and anaphase cells were determined as in Fig. 3 A. (C and D) The percentage of cells showing 3HA-Bfa1 on the dSPB, mSPB, or both (2 SPBs) for wild-type (F505) and tem1 cells expressing CNM67–TEM1 (F761) during metaphase (C) and anaphase (D). Representative images of 3HA-Bfa1 (red), tubulin (green), and DAPI (blue) are also shown. Error bars indicate SD (n = 3). Bars, 5 µm. (E) Wild-type (F496), clb2 bfa1 (F870), and tem1 cells expressing Cnm67–Tem1 in a bfa1 (F657), clb2 (F824), clb2 bfa1 (F875), or otherwise wild-type (F637) background were plated as in A. JCB • VOLUME 192 • NUMBER 4 • 2011 606 Figure 5. Constitutive loading of Tem1 onto SPBs impairs the SPOC, but not the SAC. (A) Wild-type (F496), dyn1 (F832), dyn1 kin4 (F833), CNM67–TEM1 (F637), CNM67–TEM1 dyn1 (F659), and CNM67–TEM1 dyn1 bfa1 (F835) cells were grown for 24 h in YPD at 14°C. The percentage of cells with misaligned spindles, rebudded or multi/anucleated cells, and normal cells was quantified. Error bars indicate SD (n = 3). (B–D) Wild-type (F496), Cnm67–Tem1 (F637), Gal-Kin4 (F802), and Cnm67–Tem1 Gal-Kin4 (F803) cells were grown in YPD at 25°C. (B) Cells were plated in 10-fold 607 Tem1 dynamics on spindle pole bodies regulate mitotic exit • Valerio-Santiago and Monje-Casas serial dilution spots on YPD or rich media with 2% galactose/2% raffinose (YPRG) and were grown at 25°C. The cells were also diluted at OD600 = 0.2 in YPRG and grown at 25°C. (C) A DIC image of Gal-Kin4 (F802) and Gal-Kin4 Cnm67–Tem1 (F803) cells is shown after 8 h, and compared with pictures of cells grown in YPD. Bar, 5 µm. (D) The percentage of anaphase cells was quantified through the time course. (E) Wild-type (F496), bfa1 (F533), tem1 CNM67–TEM1 (F581), tem1 CNM67–TEM1 bfa1 (F603), and tem1 BFA1–TEM1 (F615) cells were grown in YPD at 30°C and arrested in G1 with a pheromone. Cells were released into YPD with 15 µg/ml nocodazole and the percentage of rebudding was determined. An additional 7.5 µg/ml nocodazole was added every 2 h. (F) Wild-type (F496) and cells expressing Cnm67–Tem1 in a bfa1 (F657), clb2 (F824), bfa1 clb2 (F875), or otherwise wild-type (F637) background were treated as in E. constitutive loading of Tem1 onto SPBs interferes with the SPOC. To this end, we determined the effects of expressing Cnm67–Tem1 in cells lacking cytoplasmic dynein (Dyn1), a microtubule motor protein. At low temperatures, dyn1 mutants cannot efficiently position the anaphase spindle (Yeh et al., 1995), such that their viability is highly dependent on a functional SPOC. When the SPOC is impaired, dyn1 cells inappropriately exit mitosis, which leads to an accumulation of multinucleated, anucleated, and multibudded cells (Bardin et al., 2000). Although these cells account for only 6% of dyn1 cells and 2% of Cnm67–Tem1 cells growing at 14°C, this percentage increased to 55% in dyn1 cells carrying the Cnm67– Tem1 fusion (Fig. 5 A). This percentage is similar to that shown for dyn1 cells lacking Kin4, an essential component of the SPOC (Fig. 5 A), and it indicates that the constitutive presence of Tem1 on the SPBs impairs the SPOC. Failure of the SPOC in dyn1 Cnm67–Tem1 cells was not caused by an inability of the GAP to load onto SPBs, as Bfa1 still localized to SPBs (Fig. S3). Additionally, the rebudded and multi/anucleated phenotype was not increased in dyn1 bfa1 cells carrying Cnm67– Tem1 (Fig. 5 A). Kin4 promotes inhibition of mitotic exit by preventing inactivation of Bfa1-Bub2 by Cdc5 (Pereira and Schiebel, 2005). In addition, Kin4 localizes to both SPBs when the spindle is not properly aligned and blocks Tem1 loading onto this structure (D’Aquino et al., 2005). Overexpression of Kin4 also titrates Tem1 out of the SPBs and arrests cells in anaphase (D’Aquino et al., 2005). We examined the effects of constitutive targeting of Tem1 to both SPBs in cells overexpressing Kin4. Expression of Cnm67–Tem1 rescued the lethality associated with increased levels of Kin4 (Fig. 5 B). Accordingly, although overexpression of Kin4 leads to an anaphase arrest, constitutive loading of Tem1 onto both SPBs allowed cells to progress normally through the cell cycle (Fig. 5, C and D). Our results demonstrate that removal of Tem1 from the SPBs is essential for the SPOC to function. Because Bfa1 and Bub2 still load onto SPBs after SPOC activation, our results also suggest that inhibition of Tem1 by the GAP is not enough to avoid mitotic exit under these circumstances if Tem1 is not actively excluded from the SPBs. The SAC is another surveillance mechanism that requires the inhibition of MEN signaling. The SAC is triggered by unattached kinetochores, and arrests cells in metaphase by inactivation of the anaphase-promoting complex/cyclosome (APC/C; Musacchio and Salmon, 2007). MEN must also be inhibited, as cells lacking Bfa1 or Bub2 are deficient for the SAC (Fraschini et al., 1999). Cells impaired for the SAC cannot arrest the cell cycle in the presence of nocodazole (a microtubuledepolymerizing drug), which leads to inappropriate mitotic exit and the accumulation of multibudded cells. We tested whether cells carrying Cnm67–Tem1 or Bfa1–Tem1 as the only source for Tem1 could activate the SAC. Although there was a slight increase in cells showing the rebudded phenotype when compared with wildtype cells, both Cnm67–Tem1 and Bfa1–Tem1 cells maintained the metaphase arrest when treated with nocodazole (Fig. 5 E). The functionality of the SAC in cells expressing Cnm67–Tem1 was dependent on Bfa1, as deletion of BFA1 in these cells increased the rebudding to the levels of a bfa1 mutant (Fig. 5 E). Cells arrested in metaphase due to activation of the SAC show high Cdk activity. However, attenuation of the Cdk activity by CLB2 deletion in cells expressing Cnm67–Tem1 only led to a limited increase in rebudding. Furthermore, clb2 bfa1 CNM67–TEM1 cells behaved as bfa1 CNM67–TEM1 cells after nocodazole treatment (Fig. 5 F). Therefore, the differences in the functionality of the SAC and the SPOC cannot be exclusively attributed to differences in Cdk levels. Our results demonstrate that constitutive loading of Tem1 onto SPBs does not greatly disturb the SAC function, and suggest that the mechanisms by which Bfa1 restrains mitotic exit must differ for the SAC and the SPOC. Tem1 can load onto SPBs in the absence of Bfa1 Our data demonstrate that regulation of Tem1 loading onto SPBs is critical to avoid mitotic exit when the spindle is not properly aligned. But, is Tem1 loading onto the SPBs necessary for mitotic exit? Localization of Tem1 to the SPBs is dependent on both Bfa1 and Bub2 (Pereira et al., 2000), but nevertheless the bfa1 and bub2 mutants are viable. If Tem1 loading onto the SPBs is a requirement for mitotic exit, this can only be explained if Tem1 could still load to a certain level on the SPBs in the absence of Bfa1-Bub2. In fact, it has been previously shown that Tem1 can load onto the SPBs in late anaphase in a Bfa1and Bub2-independent manner (Pereira et al., 2000). Thus, we reevaluated the dependence of Bfa1 for Tem1 loading. First, we confirmed that Tem1 could load independently of Bfa1 in late anaphase in wild-type cells. We tagged Tem1 with eGFP and Bfa1 with mCherry in otherwise wild-type cells. Although most cells showed Tem1 on both SPBs during late anaphase, Bfa1mCherry was mainly asymmetric (Figs. 6 A and S4 A). The lack of signal for Bfa1-mCherry on the mSPB was not caused by differences in the level of detection for both tags, as Bfa1-mCherry could be easily detected on the mSPB in metaphase cells where the Tem1-eGFP signal was weak (Fig. 6 A). Tem1-eGFP localization mimicked that of Bfa1-mCherry during the rest of the cell cycle. Thus, our results confirm that Tem1 can load independently of Bfa1 in late anaphase and that Tem1 localization is more symmetric than that of Bfa1 in this cell cycle stage. JCB • VOLUME 192 • NUMBER 4 • 2011 614 Shou, W., J.H. Seol, A. Shevchenko, C. Baskerville, D. Moazed, Z.W. Chen, J. Jang, A. Shevchenko, H. Charbonneau, and R.J. Deshaies. 1999. Exit from mitosis is triggered by Tem1-dependent release of the protein phosphatase Cdc14 from nucleolar RENT complex. Cell. 97:233–244. doi:10.1016/S0092-8674(00)80733-3 Sikorski, R.S., and P. Hieter. 1989. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 122:19–27. Srinivasa, S.P., L.S. Bernstein, K.J. Blumer, and M.E. Linder. 1998. Plasma membrane localization is required for RGS4 function in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA. 95:5584–5589. doi:10.1073/pnas .95.10.5584 Stegmeier, F., and A. Amon. 2004. Closing mitosis: the functions of the Cdc14 phosphatase and its regulation. Annu. Rev. Genet. 38:203–232. doi:10.1146/annurev.genet.38.072902.093051 Tang, X., J.J. Punch, and W.L. Lee. 2009. A CAAX motif can compensate for the PH domain of Num1 for cortical dynein attachment. Cell Cycle. 8:3182– 3190. doi:10.4161/cc.8.19.9731 Visintin, R., and A. Amon. 2001. Regulation of the mitotic exit protein kinases Cdc15 and Dbf2. Mol. Biol. Cell. 12:2961–2974. Visintin, R., K. Craig, E.S. Hwang, S. Prinz, M. Tyers, and A. Amon. 1998. The phosphatase Cdc14 triggers mitotic exit by reversal of Cdk-dependent phosphorylation. Mol. Cell. 2:709–718. doi:10.1016/S1097-2765(00)80286-5 Visintin, R., E.S. Hwang, and A. Amon. 1999. Cfi1 prevents premature exit from mitosis by anchoring Cdc14 phosphatase in the nucleolus. Nature. 398:818–823. doi:10.1038/19775 Yeh, E., R.V. Skibbens, J.W. Cheng, E.D. Salmon, and K. Bloom. 1995. Spindle dynamics and cell cycle regulation of dynein in the budding yeast, Saccharomyces cerevisiae. J. Cell Biol. 130:687–700. doi:10.1083/ jcb.130.3.687