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Genes to Cells (2004) 9, 399-418. doi:10.1111/j.1356-9597.2004.00731.x
© 2004 Blackwell Publishing or its licensors
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The C-terminus of Bfa1p in budding yeast is essential to induce mitotic arrest in response to diverse checkpoint-activating signals

Junwon Kim, John Jeong and Kiwon Song*

Department of Biochemistry, and Institute of Life Science and Biotechnology, College of Science, Yonsei University, Seoul 120-749, Korea


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
During mitosis, genomic integrity is maintained by the proper coordination of anaphase entry and mitotic exit through mitotic checkpoints. In budding yeast, exit from mitosis is triggered by the activation of the small GTPase Tem1p. Bfa1p in association with Bub2p negatively regulates Tem1p in response to spindle damage, spindle misorientation, and DNA damage, resulting in cell cycle arrest. To delineate the Bfa1p domains that respond to distinct checkpoint signals, we constructed 13 Bfa1 deletion mutants. The C-terminal 184 amino acids of Bfa1p (Bfa1-D8391-574) contained the entire capacity of Bfa1p to generate mitotic arrest in response to spindle damage, spindle misorientation, and DNA damage. This domain was also enough to interact with the mitotic exit network proteins Tem1p, Bub2p, and Cdc5p, and to localize to the spindle pole body (SPB). Over-expression of Bfa1-D8391-574 induced late anaphase arrest as efficient as the full-length Bfa1p in a Bub2p-dependent manner. In contrast, the N-terminal portion of Bfa1p (Bfa1-D161-376) could not localize to SPB and did not block mitotic exit in response to diverse checkpoint signals. Bfa1-D161-376 interacted with Tem1p but not with Bub2p and its over-expression partially arrested cells in mitosis in the absence of Bub2p. By random mutagenesis of Bfa1-D8391-574 with hydroxylamine, we isolated a point mutant of D8, D8E438K, which interacts with both Tem1p and Bub2p but cannot respond to checkpoint signals. This mutant also showed reduced efficiency in the localization to SPB. Taken together, our study demonstrated that various checkpoint signals are transmitted to the C-terminal domain of Bfa1 (Bfa1-D8391-574) and that Bfa1p localization to SPB is necessary but not sufficient to regulate mitotic exit in response to various checkpoint signals.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Cells assure their genomic integrity during mitosis by employing mitotic checkpoints. These checkpoints monitor the assembly and orientation of the mitotic spindle and thereby ensure the equal segregation of replicated chromosomes. When the checkpoints sense defects in the microtubule cytoskeleton or detect DNA damage, they arrest the cells at metaphase and prevent them from exiting mitosis by maintaining high cyclin-dependent kinase (CDK) activity.

The regulation of the mitotic exit is best characterized in budding yeast. In this organism, mitotic exit involves a collection of genes known as the mitotic exit network (MEN) that includes TEM1, CDC15, LTE1, MOB1, DBF2, CDC5, and CDC14. These genes trigger mitotic exit once anaphase has been completed (Jaspersen et al. 1999; Morgan 1999; McCollum & Gould 2001). When mitotic exit has been triggered by an as yet unknown mechanism, the small GTPase Tem1p activates the protein kinase Cdc15p (Asakawa et al. 2001; Lee et al. 2001), which ultimately leads to the release of the protein phosphatase Cdc14p from the nucleolus (Visintin et al. 1999; Bardin et al. 2000). Cdc14p dephosphorylates the Cdh1/Hct1 protein of the anaphase-promoting complex (APC), which then becomes active and degrades Clb2p to inactivate the Clb2-Cdc28 complex (Schwab et al. 1997; Visintin et al. 1998; Zachariae et al. 1998; Jaspersen et al. 1999). The released Cdc14p also dephosphorylates and stabilizes the Clb2-Cdc28 inhibitor Sic1p and its transcription factor Swi5p, thereby promoting Clb2-Cdc28 inactivation (Knapp et al. 1996; Visintin et al. 1998). Thus, the regulation of Tem1p is important in the mitotic exit pathway, although it is not clear at the molecular level how the exit from mitosis is triggered.

It has been suggested that GTPase Tem1p is controlled both by the guanine nucleotide exchange factor (GEF) Lte1p and the two component GTPase-activating protein (GAP) complex Bub2p-Bfa1p (Morgan et al. 1999; Hoyt 2000). The GAP activity of Bub2p-Bfa1p was first inferred from work with Schizosaccharomyce pombe. byr4p and cdc16p of S. pombe are homologues of Bfa1p and Bub2p and form a two-component GAP for the GTPase activity of a Tem1p homologue, spg1p, in vitro (Furge et al. 1998). A recent report has shown that Bfa1p and Bub2p also act as a two-component GAP for Tem1p activity in vitro (Geymonat et al. 2002). Tem1p binds Bfa1p and Bub2p on the cytoplasmic side of the spindle pole body (SPB) whereas Lte1p is associated with the cortex of the bud (Bardin et al. 2000; Pereira et al. 2000). This spatial separation of the elements regulating Tem1p activity suggests that Tem1p remains inactive in its GDP-bound form by Bub2p-Bfa1p at the SPB during most of the cell cycle and that Lte1p converts Tem1p to the GTP-bound active form when the SPB enters the daughter cell at the execution of anaphase (Bardin et al. 2000; Pereira et al. 2000).

Bfa1p and Bub2p prevent the untimely activation of mitotic exit in the absence of proper nuclear division by monitoring defects in the migration of the SPB into the bud, as well as monitoring for astral microtubule structures and DNA damage (Bloecher et al. 2000; Wang et al. 2000; Pereira et al. 2001). These data suggest that Bfa1p and Bub2p function as a universal checkpoint in response to various checkpoint signals to avoid improper mitotic exit. The phosphorylation of Bfa1p by the polo-like kinase Cdc5p antagonizes the ability of Bfa1p to permit mitotic exit (Hu et al. 2001; Geymonat et al. 2003). However, it is still not well understood how different mitotic checkpoint signals are integrated into Bfa1p and Bub2p for the regulation of Tem1p. In addition, deletion of LTE1 has little effect on the timing of mitotic exit at 30 °C, and several mutant cells in which the SPB does not migrate into bud or the neck can exit mitosis inappropriately to produce binucleated and anucleated cells, suggesting that the proposed model does not fully explain the mechanism of Tem1p regulation (Adames et al. 2001). Recently, Bfa1p was shown to regulate Tem1p to control mitotic exit in the absence of Bub2p (Ro et al. 2002). These data strongly suggest that Bfa1p is a key regulator of Tem1p in response to various mitotic checkpoint signals and that it may regulate Tem1p by an additional mechanism apart from its activity in the two-component GAP with Bub2p.

In this study, we examined the functional domains of Bfa1p that respond to several distinct checkpoint signals by analysing 13 Bfa1p deletion mutants. We show that the function of Bfa1p for mitotic arrest is concentrated on its C-terminal 184 residues. Our mutagenesis study of the C-terminal 184 residues also suggest that Bfa1p localization to the SPB is necessary but not sufficient to induce mitotic arrest in response to the checkpoint-activating signals.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Bfa1p contains 574 amino acids and shows limited homology to the S. pombe byr4p in its C-terminal region, including the direct repeats and sequences surrounding these repeats (Song et al. 1996; Lee et al. 1999; Fig. 1). Bfa1p is required to prevent mitotic exit and is a target of regulation in response to spindle damage, spindle misorientation, and DNA damage (Li 1999; Bloecher et al. 2000; Wang et al. 2000; Hu et al. 2001; Pereira et al. 2001). bfa1{Delta} is defective in reacting to these checkpoint-activating damages and continues the cell cycle, resulting in re-budding and a sharp decrease in viability. To delineate the functional domains of Bfa1p that negatively regulate mitotic exit in response to diverse checkpoint signals, we constructed 13 Bfa1 deletion mutants and examined the ability of each mutant to induce mitotic arrest by checkpoint-activating signals, as summarized in Fig. 1.



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  		Figure 1  Summary of the functional domains of Bfa1p. Schematic diagrams illustrate the sequence alignment of Bfa1p, the S. pombe homologue byr4p, and the 13 Bfa1p deletion mutants used in this study. The arrows and the boxes, respectively, represent the direct repeats (residues 326–371 and 391–437) and the regions of significant sequence similarity (residues 302–340 and 501–532) between Bfa1p and byr4p, as identified by BLAST and reported by Song et al. (1996). The expression of Bfa1p and its 13 deletion mutants was verified and the ability of each mutant to maintain mitotic arrest in response to spindle damage, spindle misorientation, and DNA damage as well as to localize to the SPB and to induce mitotic arrest by over-expression was examined. In the over-expression (++) indicates the same degree of mitotic arrest as full-length Bfa1 (+) less mitotic arrest than full-length Bfa1, and (–) the same lack of mitotic arrest as the vector control. In other assays (+) or (–) indicates the ability to arrest mitosis in response to each signal and to localize on the SPB or not, respectively. ND, not determined.

 
Bfa1-D8391-574, the 184 amino acids in the C-terminus, is sufficient to arrest mitosis exit in response to spindle damage

We first identified the domain of Bfa1p that is required for mitotic arrest by spindle damage. The 13 BFA1 deletion mutants in low copy pCEN plasmid were tested for their ability to restore the benomyl sensitivity of bfa1{Delta} cells. The expression of each truncated mutant was verified prior to the functional assays of each mutant (Fig. 2A, right panel; data not shown). The full-length BFA1, BFA1-D8391-574, and BFA1-D1302-574 that includes the residues of D8, entirely restored the benomyl sensitivity of bfa1{Delta}, but BFA1-D161-376 could not (Fig. 2A). To confirm that BFA1-D8391-574 is sufficient to induce mitotic arrest by spindle damage, we examined the prevalence of multiple buds and the viability of bfa1{Delta} cells containing each BFA1 deletion mutant when mitotic spindle formation was inhibited by nocodazole (Fig. 2B,C). Consistent with the data shown in Fig. 2A, the full-length BFA1 and D8391-574 suppressed the re-budding and cell cycle progression of nocodazole-treated bfa1{Delta} cells, while D161-376 and the vector control did not (Fig. 2B). D8391-574 also improved the viability of nocodazole-treated bfa1{Delta} cells as the full-length BFA1 (Fig. 2C).



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  		Figure 2  The C-terminal 184 amino acids of Bfa1p, Bfa1-D8, bear the ability of Bfa1p to induce M arrest in response to spindle damage. (A) BFA1 deletion mutants were tested for their ability to induce mitotic arrest in bfa1{Delta} cells treated with benomyl. bfa1{Delta} cells (YSK8) carrying each BFA1 deletion mutant in a low copy pCEN plasmid were grown to equivalent densities, serially diluted 10-fold, and spotted on to either YPDA plates (left) or YPDA plates containing 10 µg/mL benomyl (right). The plates were incubated at 25 °C either for 2 days (–benomyl) or 3 days (+benomyl) for growth. Each truncated mutant was expressed as shown Bfa1p, Bfa1p-D8, and Bfa1p-D16 in the Western blot. (B, C) BFA1 deletion mutants were tested for their ability to (B) reduce the new bud formation and (C) improve the viability of nocodazole-treated bfa1{Delta} cells. (B) Logarithmically growing bfa1{Delta} cells (YSK8) carrying D8 or D16 in low copy pCEN plasmids were shifted to YPDA containing 15 µg/mL nocodazole at 25 °C. The number of cells with more than one bud was counted every hour for 6 h after fixing and briefly sonicating the cells. 300 cells were counted at each time point and the percentage of cells with more than one bud was calculated relative to the total number of counted cells. (C) Aliquots of cells removed for assays in (B) were diluted, sonicated, plated on YPDA for 24 h at 25 °C, and scored for colony formation. The percentage of viable cells was calculated relative to the number of viable cells at time 0. The average of three independent experiments was presented in (B, C). (D) BFA1 deletion mutants were tested for their ability to induce mitotic arrest in ask1-2 bfa1{Delta} double mutant. Logarithmically growing ask1-2 bfa1{Delta} cells (YSK16) carrying BFA1-D8 and D16 in low copy pCEN plasmids and ask1-2 bub2{Delta} (YSK17) cells were arrested with {alpha}-factor at 25 °C and released at 37 °C. Cells were taken every 45 min and their DNA contents were analysed by flow cytometry.

 
Ask1p is a component of the budding yeast Duo1p-Dam1p-Dad1p complex that functions to establish and maintain the bipolar attachment of microtubules to sister kinetochores (Janke et al. 2002). Thus, the ask1-2 temperature-sensitive mutant shows segregation of DNA masses without the separation of sister chromatids and activates the spindle checkpoint at the non-permissive temperature, to arrest cells with undivided nuclei and a short spindle (Hu et al. 2001; Li et al. 2002). We examined whether Bfa1p is also involved in the mitotic arrest in response to the defects of bipolar attachment of microtubules to kinetochore in ask1 mutant. In contrast to ask1-2, the ask1-2 bfa1{Delta} double mutant lacks the mitotic arrest due to the deletion of BFA1 and proceeds with the cell cycle at the restrictive temperature, resulting in G1 and G2 DNA content (Fig. 2D). Similarly, ask1-2 bub2{Delta} also progresses the cell cycle at the restrictive temperature (Fig. 2D). We then assessed whether BFA1-D8391-574, which is sufficient to induce mitotic arrest by nocodazole, will also activate the spindle checkpoint in the ask1-2 bfa1{Delta} double mutant. We measured DNA content and found that full-length BFA1 and D8391-574 fully recovered the ability of ask1-2 bfa1{Delta} to induce mitotic arrest in response to spindle damage, while D161-376 and the vector control did not alter the cell cycle progression of ask1-2 bfa1{Delta} cells at all (Fig. 2D). These observations demonstrate that Bfa1p-D8391-574 can respond to various spindle damages including nocodazole treatment and the mutation of ASK1, indicating that this domain encompasses the spindle checkpoint functions of Bfa1p in response to diverse spindle damages.

Bfa1-D8391-574 is sufficient to induce mitotic arrest by spindle misorientation

Bfa1p prevents mitotic exit when the mitotic spindle is misoriented by a defect in the pathways that have been reported to orientate the spindle into the neck (Bloecher et al. 2000; Pereira et al. 2001; J. Kim and K. Song, unpublished observation). They are two partially redundant mechanisms, namely, the dynein/dynactin-dependent sliding of microtubules along the bud cortex, and the Kar9p- and Bim1p-dependent capture of microtubules at the bud cortex (Adames et al. 2001; Korinek et al. 2000). When BFA1 was missing, bim1{Delta} or kar9{Delta} cells show reduced survival at 23 °C, and die at 37 °C suggesting that BFA1 is important for the survival of cells with astral microtubule defects (Pereira et al. 2001). In addition, dyn1{Delta} bfa1{Delta} and dyn1{Delta} bub2{Delta} double mutant cells progress with mitosis, while, in contrast, dyn1{Delta} cells are arrested at mitosis (Bloecher et al. 2000; J. Kim and K. Song, unpublished observaiton). Thus, we delineated the domain of Bfa1p involved in the checkpoint function of Bfa1p in response to spindle misorientation by testing the ability of the 13 BFA1 deletion mutants to rescue the lethality of bim1{Delta} bfa1{Delta} at 35 °C. Full-length BFA1, D8391-574, and D1302-574 entirely reversed the lethality of bim1{Delta} bfa1{Delta}, unlike the vector control and D161-376 (Fig. 3A). When we examined the number of cells with multiple buds and the spindle orientation in bim1{Delta} bfa1{Delta} cells transformed with D8391-574 or D161-376, we found that, like full-length BFA1, D8 suppressed the new bud formation and cell cycle progression of bim1{Delta} bfa1{Delta}, while D16 and the vector control had no such effect (Fig. 3B,C). The bim1{Delta} bfa1{Delta} cells transformed with the full BFA1 or D8391-574 were arrested in mitosis with misoriented spindles, as is bim1{Delta}, but the cells with D161-376 formed new buds despite the misaligned spindle and missegregated DNA (Fig. 3C). Thus, D8391-574 contains the checkpoint functions of Bfa1p needed for cells to survive the astral microtubule defects resulting from the deletion of BIM1.



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  		Figure 3  Bfa1-D8 bears the ability of Bfa1p to generate M arrest in response to spindle misorientation. (A–C) BFA1 deletion mutants were tested for their ability to (A) rescue the synthetic lethality and (B, C) suppress the new bud formation of bim1{Delta} bfa1{Delta} cells (YSK19). (A) bim1{Delta} bfa1{Delta} cells carrying each BFA1 deletion mutant in a low copy pCEN plasmid were serially diluted 10-fold, spotted on to YPDA plates, and incubated either at 25 °C for 3 days (left) or at 35 °C for 5 days (right). (B, C) Cells were arrested with 0.2 M hydroxyurea at 25 °C and released at 35 °C. (B) Cells were collected at the indicated time points after the release and the number of cells with more than one bud was counted. Three hundred cells were counted at each time point and the percentage of cells with more than one bud was calculated relative to the total number of counted cells. The average of three independent experiments is presented. (C) 6 h after release, the cell phenotypes were observed by fluorescence microscopy. DNA (left) and GFP-microtubules (right) are shown. Bar, 10 µm. (D, E) BFA1 deletion mutants were tested for their ability to block the new bud formation of dyn1{Delta} bfa1{Delta} (YSK21). (D) dyn1{Delta} bfa1{Delta} cells carrying each BFA1 deletion mutant in a low copy pCEN plasmid were grown to mid-log phase, arrested with 0.2 M hydroxyurea at 30 °C for 3–4 h, and released in YPDA medium containing 5 µg/mL {alpha}-factor at 16 °C for 6 h. The number of anucleate and multinucleate cells was counted after stained with DAPI and the percentage of anucleate and multinucleate cells was calculated relative to the total number of counted cells. The average of three independent experiments is presented. (E) Phenotypes of the cells grown asynchronously at 16 °C for 24 h were observed after stained with DAPI. Bar, 10 µm.

 
The dyn1{Delta} bfa1{Delta} double mutant undergoes mitosis despite a misaligned spindle and therefore produces multinuclear mother cells and anucleate cells that often contain an extra bud (Fig. 3E). This cell cycle progression is more obvious when the cells are incubated at 16 °C (J. Kim and K. Song, unpublished observation). To define the domain(s) of Bfa1p that maintain(s) the checkpoint function of Bfa1p to arrest at mitosis in response to spindle misorientation induced by lack of dynein, we tested each of the 13 BFA1 deletion mutants for its ability to suppress the cell cycle progression of dyn1{Delta}bfa1{Delta}. We counted the number of anucleate and multinucleate cells and cells forming an extra bud. We found these numbers were decreased by equivalent extents in dyn1{Delta} bfa1{Delta} cells transformed with D8391-574 and full-length BFA1 (Fig. 3D). In contrast, D161-376 and the vector control were unable to arrest dyn1{Delta} bfa1{Delta} cells in mitosis (Fig. 3D,E). Thus, D8391-574 appears to encompass the domain responsible in Bfa1p for sensing and responding to a misaligned spindle and improper chromosome segregation.

Bfa1-D8391-574 is sufficient to arrest mitosis in response to DNA damage

Cells with the temperature-sensitive cdc13-1 mutation accumulate single-stranded DNA at non-permissive conditions and activate the DNA-damage checkpoint (Garvik et al. 1995). Bfa1p is required to inhibit the mitotic exit in response to the DNA damage induced by cdc13-1 mutation (Wang et al. 2000; Hu et al. 2001). At the restrictive temperature, the cdc13-1 bfa1{Delta} double mutant shows re-budding and re-replication due to cell cycle progression, whereas cdc13-1 cells arrest with a large bud and fail to re-replicate (Wang et al. 2000). To delineate the domain of Bfa1p involved in DNA damage-induced mitotic arrest, the 13 BFA1 deletion mutants were transformed into cdc13-1 bfa1{Delta} cells and tested for their ability to repress the new bud formation of cdc13-1 bfa1{Delta} at the non-permissive temperature. When cells with more than one bud were counted, BFA1-D8391-574 and full-length BFA1 completely inhibited new bud formation, unlike D161-376 and the vector control (Fig. 4A,B). cdc13-1bfa1{Delta} cells transformed with D8 or full-length BFA1 arrested with an enlarged bud, verifying that D8 is able to arrest the cell cycle progression of cdc13-1bfa{Delta} (Fig. 4B). Confirming this is that when we measured DNA content, we found BFA1-D8 and full-length BFA1 arrested cdc13-1bfa1{Delta} cells with G2 DNA contents (Fig. 4C). In contrast, some cdc13-1bfa1{Delta} cells transformed with D16 or the vector control underwent re-replication and therefore showed higher a DNA content than G2 (Fig. 4C). These results demonstrate that Bfa1p-D8391-574 is sufficient to block mitotic exit in response to DNA damage.



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  		Figure 4  Bfa1p-D8 bears the ability of Bfa1p to induce M arrest in response to DNA damage. (A–C) BFA1 deletion mutants were tested for their ability to suppress the new bud formation of cdc13-1 bfa1{Delta} (YSK23). cdc13-1 bfa1{Delta} cells carrying each BFA1 deletion mutant in a low copy pCEN plasmid were grown to mid-log phase at 25 °C arrested with {alpha}-factor and released at 35 °C for 6 h. (A) Cells were collected every hour and the number of cells with more than one bud was counted. 300 cells were counted at each time point and the percentage of cells with more than one bud was calculated relative to the total number of counted cells. The average of three independent experiments is presented. (B) 6 h after the release, cells were stained with DAPI and the phenotypes were observed by fluorescence microscopy. Bar, 10 µm (C) Aliquots of cells removed for the assay in (A) were analysed by flow cytometry.

 
Only D8 can aggravate the defect of Cdc5p mutations that is suppressed when Bfa1p is deleted

Bfa1p activity in response to multiple checkpoint signals is regulated by its phosphorylation. The phosphorylation of Bfa1p by the polo-like kinase Cdc5p antagonizes the ability of Bfa1p to promote mitotic exit (Hu et al. 2001; Geymonat et al. 2003). One would expect therefore that BFA1 and CDC5 interact genetically together. Supporting this is the observation that deletion of BFA1 has been reported to rescue the mitotic exit defect of cdc5 mutant alleles. For example, the cdc5-2 mutant allele inhibits Bfa1p phosphorylation and mitotic exit at the restrictive temperature, but this phenotype is suppressed by the deletion of BFA1 (Hu et al. 2001). In addition, loss of BFA1 alleviates the mitotic exit defect of the cdc5-1 mutation, although the cdc5-1 mutant allele phosphorylates Bfa1p (Ro et al. 2002). Both the cdc5-1 and cdc5-2 mutant alleles showed a growth defect in the restrictive temperature that is suppressed in cdc5-1 bfa1{Delta} and cdc5-2 bfa1{Delta} (Fig. 5A, right panels). We tested therefore whether D8391-574 or D161-376 could reduce the growth of cdc5-1 bfa1{Delta} and cdc5-2 bfa1{Delta} cells in the restrictive temperature. cdc5-1 bfa1{Delta} and cdc5-2 bfa1{Delta} cells transformed with D8391-574 or full-length BFA1 in low copy pCEN plasmids and transferred to the non-permissive temperature showed a reappearance of the growth defects (Fig. 5A). In contrast, D161-376 and the vector control did not affect the growth of the double mutants (Fig. 5A).



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  		Figure 5  Bfa1-D8 restores the mitotic exit defects of Cdc5p mutations that are suppressed in cdc5bfa1{Delta}. (A) Mitotic exit defects of both cdc5-1 and cdc5-2 are alleviated by the deletion of BFA1. Bfa1-D8 and D16 were tested for their ability to reverse this effect. cdc5-1 bfa1{Delta} (KLY2454, top) and cdc5-2 bfa1{Delta} (Y1151, bottom) cells carrying each BFA1 deletion mutant in a low copy pCEN plasmid were grown to mid-log phase at 25 °C, serially diluted 10-fold, and spotted on to YPDA plates. The plates were incubated either at 25 °C for 2 days (left) or at 35 °C for 1 day (right). (B) The ability of Bfa1 deletion mutants to interact physically with Cdc5p was analysed by yeast two-hybrid assays. ß-galactosidase activity was measured and plotted for each Bfa1 mutant in the yeast two-hybrid assays. The grey and black bars, respectively, represent ß-galactosidase activity with and without the induction of Cdc5p expression. The expression of Cdc5p as well as Bfa1p, Bfa1p-D8, and Bfa1p-D16 were shown in the Western blots (right panel).

 
Hu et al. (2001) found 11 Cdc5p-dependent phosphorylation sites on Bfa1p. Of these, D161-376 has nine and D8391-574 has two. This suggests that both domains must physically interact with Cdc5p to be phosphorylated. However, we found that only D8391-574 aggravates the mitotic exit defect of the cdc5 mutant alleles that is suppressed by the deletion of BFA1 (Fig. 5A). We thus delineated the domains of Bfa1p that physically interact with Cdc5p by yeast two-hybrid and co-precipitation assays. In yeast two-hybrid assays, both D8391-574 and D161-376 strongly interacted with Cdc5p (Fig. 5B). However, we hardly detected the co-precipitation of Bfa1-D8 or D16 with Cdc5-3HA (data not shown), suggesting that Bfa1p interacts with Cdc5 transiently.

D8391-574 and D161-376 are two independent domains that induce mitotic arrest when over-expressed

In this study we showed that D8391-574 is sufficient to arrest mitosis in response to spindle damage, spindle misorientation, and DNA damage. This domain was also sufficient to genetically interact with and to be regulated by Cdc5p. Thus, we compared the ability of each mutant to induce mitotic arrest by over-expressing it in bfa1{Delta} cells (Fig. 1) and examined whether D8391-574 is the only domain to induce mitotic arrest by over-expression. When over-expression was induced for 4 h, Bfa1-D8391-574 could arrest cells in anaphase as well as the full-length Bfa1 (Fig. 6A). In addition, Bfa1-D161-376 could induce mitotic arrest (Fig. 6A). However, the number of cells arrested in anaphase by over-expressing the mutants bearing residues 225–376 was approximately 70% of D8391-574 or the full-length Bfa1 (Figs 1 and 6A). These observations indicate that D8391-574 and D161-376 are two independent domains that induce mitotic arrest when over-expressed and that D8391-574 was as capable of inducing mitotic arrest as the wild-type Bfa1p. Based on two independent functional domains for mitotic arrest as well as the differences in their ability to respond diverse checkpoint signals, Bfa1p could be bipartite into the N-terminal 376 amino acids (D161-376) and the C-terminal 184 amino acids (D8391-574).




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  		Figure 6  Bfa1 can be bipartite into the N-terminal 376 amino acids (Bfa1-D16) and the C-terminal 184 amino acids (Bfa1-D8) that, respectively, induce mitotic arrest by over-expression. (A) The Bfa1 deletion mutants were over-expressed in bfa1{Delta} cells (YSK8) and the ability of each mutant to induce mitotic arrest was examined. The percentage of cells in anaphase was plotted when each of the 13 Bfa1 deletion mutants was over-expressed in bfa1{Delta} under the GAL10-1 promoter for 4 h. More than 500 cells were counted for each mutant and cells in anaphase were scored after staining with DAPI. The data of the full-length Bfa1, D8, and D16 are presented with the vector control. (B, C) The ability of Bfa1-D8 and D16 to interact physically with (B) Tem1p or (C) Bub2p was investigated by yeast-two-hybrid (left) and co-precipitation (right) assays. ß-galactosidase activity was measured and plotted in the yeast two-hybrid assays. The grey and black bars, respectively, represent ß-galactosidase activity with and without the induction of Tem1p or Bub2p expression. As shown in Western blots (middle) (B) Tem1p and (C) Bub2p as well as Bfa1p, Bfa1p-D8, and Bfa1p-D16 were expressed when induced. For co-precipitation assays, GFP-tagged Bfa1p-D8 and D16 with GFP vector control, and 3xHA-tagged Tem1p or Bub2p under GAL10-1 promoter were, respectively, expressed in bfa1{Delta} (YSK8). Each lysate of separately expressed Bfa1-D8 and D16 tagged with GFP and Tem1p or Bub2p tagged with 3xHA was mixed together and purified with anti-HA and protein A-agarose. Each lysate and co-precipitates with anti-HA were, respectively, blotted with anti-GFP and anti-HA antibodies. The GFP fusion of each Bfa1 deletion mutant of expected size was marked with arrowhead. (D) The percentage of cells in anaphase was scored when D8 and D16 were over-expressed in bfa1{Delta} bub2{Delta} cells (YSK26) under the GAL10-1 promoter for 4 h. Full-length BFA1 and vector only were used as controls. More than 500 cells of each mutant were counted after stained with DAPI. (E) The growth arrest by D8 and D16 over-expression was reversed by the over-expression of Tem1p. bfa1{Delta} (YSK8) was transformed with pCEN-PGAL-BFA1-GFP, D8, and D16, and/or pCEN-PGAL-TEM1-HA. Each transformant with the combinations of plasmids was spotted on to either glucose or galactose containing medium to examine the growth. The expression of full-length Bfa1, D8, and D16 as well as Tem1p in each transformant was shown in the bottom Western blot. (F) The localization of Cdc14p in bfa1{Delta} cells in which mitotic arrest is induced by over-expressing Bfa1-D8, D16, and full-length Bfa1. Bfa1-D8, D16, and full-length Bfa1 were over-expressed for 4 h in bfa1{Delta} cells in which chromosomal CDC14 has been tagged with 5 x GFP (YSK11). DNA is shown by DAPI staining (left) and Cdc14p by GFP tagging (right). Bar, 10 µm.

 
Since over-expression of Bfa1p inhibits mitotic exit by its binding and negatively regulating Tem1p, we delineated the domains of Bfa1p responsible for binding to Tem1p by yeast two-hybrid and co-precipitation assays. As both D8391-574 and D161-376 induced mitotic arrest by over-expression, they may contain the minimal domains involved in Tem1p binding. Thus, we examined the binding of D8391-574 and D161-376 to Tem1p after verifying the expression of Tem1p as well as the full-length Bfa1, D8, and D16. Co-precipitation assays and yeast two-hybrid assays revealed that both D8391-574 and D161-376 do bind with Tem1p (Fig. 6B). These experiments confirmed that both the C-terminal 184 amino acids and the N-terminal 376 amino acids of Bfa1p contain the minimal domains for Tem1p binding (Fig. 6B). The observation that both D8 and D16 bind to Tem1p also suggests that the ability of these two domains to induce mitotic arrest upon their over-expression is mediated by their interactions with Tem1p.

It is likely that Bfa1p negatively regulates mitotic exit at least partly by associating with Bub2p to form a two component GAP. Thus, we verified the expression of Bub2p as well as the full-length Bfa1, D8, and D16, and delineated the Bub2p binding domain of Bfa1p by yeast two-hybrid and co-precipitation assays (Fig. 6C). We found that only D8391-574 interacts with Bub2p as the full-length, while D161-376 could not interact with Bub2p (Fig. 6C). This observation suggests that over-expressed D161-376 interacts with Tem1p and partially induces mitotic arrest without binding to Bub2p. To test whether the mitotic arrest resulting from D8 over-expression depends on Bub2p while D16 induce mitotic arrest regardless of Bub2p, we over-expressed D8391-574 and D161-376 in bfa1{Delta}bub2{Delta} and examined the cells in the anaphase. As expected from the binding of Bub2p to D8 but not to D16, over-expressing D8 or the vector control in the absence of BUB2 did not induce mitotic arrest but over-expression of D16 or the full BFA1 continued to partially induce mitotic arrest (Fig. 6D). These observations demonstrated that the mitotic arrest by D8 over-expression is Bub2p-dependent, while D16 over-expression induces the arrest in the absence of Bub2p.

Since both D8391-574 and D161-376 block mitotic exit by over-expression and interact with Tem1p, we also investigated whether these domains inhibit mitotic exit by binding and regulating Tem1p. Thus, we examined whether Tem1p over-expression overrides the arrest of cell growth by the over-expression of D8391-574 and D161-376. The over-expression of Tem1p was confirmed in the cells expressing full-length Bfa1, D8, and D16, respectively (Fig. 6E, lower panel). The mitotic arrest by the over-expression of D8391-574 and D161-376 as well as the full-length Bfa1p was reversed by Tem1p over-expression (Fig. 6E). These observations suggest that the inhibition of mitotic exit imposed by the over-expression of D8391-574 and D161-376 depend on Tem1p, while D8 over-expression induces the arrest in a Bub2p-dependent manner.

Considering that Cdc14p is released from the nucleolus when mitotic exit is triggered (Shou et al. 1999; Visintin et al. 1999; Bardin et al. 2000) and that Bfa1p negatively regulates mitotic exit upstream of Cdc14p, over-expression of Bfa1p should block the release of Cdc14p from the nucleolus, thereby inhibiting mitotic exit. To verify the mitotic arrest by over-expression of D8391-574 and D161-376, we also examined the block of Cdc14p release from the nucleolus. As shown in Fig. 6F, Cdc14p was confined in the nucleolus of the cells over-expressing D8391-574, D161-376, and full-length Bfa1p. In contrast, Cdc14p was spread in and out of the nucleus in cells that over-expressed the vector only. Thus, the two Bfa1p domains D8391-574 and D161-376 independently inhibit mitotic exit when they are over-expressed.

Only Bfa1-D8391-574 is necessary for Bfa1p localization to the SPB

The localization of Bfa1p to SPB has been reported to be dependent on Bub2p, and vice versa (Pereira et al. 2000). However, our observations above indicate that the two Bfa1p domains D8391-574 and D161-376 both induce mitotic arrest although only D8 interacts directly with Bub2p. Only D8 also functions to arrest mitosis in response to diverse checkpoint-activating damages. To determine whether the localization of Bfa1p to the SPB is directly correlated with its checkpoint functions, we delineated the domain in Bfa1p that drives its localization to the SPB by expressing the 13 Bfa1 deletion mutants as GFP-fusions (Figs 1 and 7A). We also verified that each Bfa1p deletion mutant tagged with GFP is expressed by performing Western blots using an anti-GFP antibody. Each deletion mutant of the expected size was detected (Fig. 7B, data not shown). Only D8391-574 as well as the full-length Bfa1 localized to the SPB, and D161-376 was not detected at the SPB (Fig. 7A). The localization of D8391-574 to the SPB was also verified using a SPB marker, Spc42p. Co-localization of D8 and Spc42p was observed in YSK40 in which YFP-tagged BFA1-D8 and CFP-tagged SPC42 were integrated in bfa1{Delta} (YSK40). Bfa1-D8 expressed under the own promoter of BFA1 was enough to localize on the SPB (Fig. 7C). Co-localization of YFP-tagged D8391-574 to the SPB with CFP-tagged Spc42p was verified in the superimposed enlarged image, as shown in Fig. 7C. Thus, D8391-574, which contains the Bub2p interacting domain, also bears the capacity of Bfa1p to localize to the SPB, suggesting that the localization of Bfa1p to the SPB is directly correlated with its ability to regulate the mitotic exit in response to various checkpoint signals. Furthermore, the observation with D16 suggests that the localization of Bfa1p to the SPB is not required to induce mitotic arrest by over-expression.



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  		Figure 7  Bfa1-D8 bears the SPB localization activity of Bfa1p. (A) Localization of the Bfa1 deletion mutants to the SPB in bfa1{Delta} cells (YSK8). The data of the full-length Bfa1, D8, and D16 are presented with the vector control. GFP-tagged D8, D16, full-length Bfa1, and the GFP vector only were expressed in bfa1{Delta} cells under the GAL10-1 promoter for 4 h. DNA is shown by DAPI staining (left) and Bfa1-GFP by GFP tagging (right). Bar, 10 µm. (B) The expression of GFP-tagged full-length Bfa1, D8, and D16 in bfa1{Delta} (YSK8) is shown by anti-GFP immunoblot. Protein extracts were prepared from bfa1{Delta} cells in which the expression under the GAL10-1 promoter of GFP-tagged Bfa1, D8, and D16 was induced for 4 h. The GFP fusion of Bfa1 deletion mutants of expected size was marked with arrowhead. (C) Localization of Bfa1-D8 to the SPB was verified by its co-localization with a SPB marker Spc42p. The right panel shows the merged image of Spc42-CFP and Bfa1-D8-YFP of BFA1-D8-YFP SPC42-CFP bfa1{Delta} (YSK40). Bar, 10 µm.

 
The mutant of Bfa1-D8, D8E438K, is deprived of the checkpoint function of D8

We have so far presented that Bfa1-D8391-574 bears the ability of Bfa1p to localize to the SPB and to regulate the mitotic exit in response to various checkpoint signals, while Bfa1-D161-376 that does not localize to the SPB cannot react to the mitotic checkpoint signals despite of its binding to Tem1p. To understand the correlation of the essential checkpoint functions of D8391-574 and its localization to the SPB, we screened the mutations of Bfa1-D8391-574 that lose the checkpoint activity and examined the localization of a checkpoint-deficient mutant. We isolated a point mutation, D8E438K, by random mutagenesis of D8 with hydroxylamine, as described in Experimental procedures. We verified the loss of checkpoint function in D8E438K by examining the restore of the benomyl sensitivity in bfa1{Delta} cells, after D8E438K was integrated in bfa1{Delta} (YSK39). Both Bfa1p-D8 and D8E438K were expressed as shown in the Western blot, but the benomyl sensitivity of bfa1{Delta} was entirely restored by BFA1-D8391-574 but not by D8E438K (Fig. 8A), demonstrating that D8E438K could not respond to spindle damage any more. Consistent with the lack of checkpoint function, D8E438K no longer arrested cells in mitosis when over-expressed (data not shown). We then examined whether Bfa1-D8E438K could localize to the SPB as D8, when expressed as a GFP-fusion. As shown in Fig. 8B, Bfa1-D8E438K as well as D8 localized to the SPB, although the efficiency of localization to SPB is slightly reduced in D8E438K. Since the localization of Bfa1p to SPB has been reported to rely on Bub2p and vice versa (Pereira et al. 2000), we also examined the interaction of Bfa1-D8E438K with Bub2p by co-precipitation. As expected from its localization to SPB, D8E438K interacted with Bub2p (Fig. 8C). Furthermore, D8E438K interacted with Tem1p despite of the loss of its checkpoint function (Fig. 8C). These observations demonstrate that the mutagenized glutamine residue is critical for the checkpoint functions of Bfa1p and strongly suggest that the localization of Bfa1p to the SPB as well as its binding to Bub2p and Tem1p are necessary but not sufficient for the essential checkpoint functions of Bfa1p.



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  		Figure 8  The mutant of Bfa1-D8391-574, D8E438K, is deprived of the checkpoint function of D8 for MEN regulation. (A) BFA1-D8 mutant, D8E438K, lost its ability to induce mitotic arrest in bfa1{Delta} cells treated with benomyl. bfa1{Delta} cells (YSK8) integrated with either BFA1-D8 (YSK38) or D8E438K (YSK39) were grown to equivalent densities, serially diluted 10-fold, and spotted on to either YPDA plates (left) or YPDA plates containing 10 µg/mL benomyl (right). The plates were incubated at 25 °C either for 2 days (–benomyl) or 3 days (+benomyl) for growth. The similar expression of D8 and D8E438K was shown in a bottom Western blot. (B) The localization of Bfa1-D8E438K to the SPB was observed in bfa1{Delta} cells. D8E438K-GFP and D8-GFP were expressed in bfa1{Delta} cells (YSK8) under the GAL10-1 promoter for 4 h in the medium containing 2% galactose and 0.15% glucose to allow limited expression of fusion proteins. The expression of D8 and D8E438K was verified as shown in the Western (bottom). DNA is shown by DAPI staining (left) and Bfa1-GFP by GFP tagging (right). Bar, 10 µm. Efficiency of the SPB localization of D8E438K was compared with D8 by counting cells in which Bfa1 localizes on SPB and plotting the percentage. Total 200 cells were counted. (C) Bfa1-D8E438K, which is deprived of the checkpoint function of D8, interacts with Bub2p and Tem1p by co-precipitation. 3 x HA-tagged D8E438K and GST-tagged Bub2p and Tem1p under GAL10-1 promoter were, respectively, introduced into bfa1{Delta} (YSK8), separately expressed, and verified by immunoblots (upper panels). Each lysate of separately expressed 3 x HA-tagged Bfa1-D8E438K and GST-tagged Tem1p or Bub2p was mixed together, purified with glutathione-agarose (Sigma), and blotted with anti-GST and anti-HA antibodies (bottom panels).

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
In this paper, we defined the functional domains of Bfa1p that are involved in preventing mitotic exit in response to various checkpoint signals. Our experiments revealed that there are two separate domains in Bfa1p that can induce mitotic arrest when over-expressed. One is contained in the C-terminal residues 391–574, the other in the N-terminal residues 1–376. However, we showed that the entire capacity of Bfa1 to sense spindle damage, spindle misorientation, and DNA damage is contained in the C-terminal 184 amino acids (D8391-574). Bfa1-D8391-574 also bears the SPB-localizing activity of Bfa1 as well as its ability to interact with the mitotic exit network proteins Tem1 p, Bub2p, and Cdc5p. Over-expression of D8391-574 induced late anaphase arrest in a Bub2p-dependent manner, and was relieved by simultaneous over-expression of Tem1p, suggesting that D8 bind and negatively regulate Tem1p in a Bub2p-dependent means. These data strongly suggest that diverse checkpoint signals are transmitted to D8391-574, that sensing the checkpoint signals by D8391-574 is likely correlated with its localization to the SPB, and that this domain down-regulates the activity of Tem1p GTPase to induce mitotic arrest in a Bub2p-dependent way probably by acting in a two-component GAP with Bub2p. Our observations are consistent with the report that the C-terminal fragment of Bfa1p encompassing residues 391–574 binds to Bub2p and that this complex has GAP activity in vitro (Geymonat et al. 2002). Our study of the functional domains of Bfa1p in S. cerevisiae is consistent with the functional domains of byr4 in S. pombe. In byr4, the C-terminal 177 amino acids (479–655) are enough to mediate both the localization to SPB and the Bub2p homologue cdc16-dependent spg1 GAP activity (Furge et al. 1999; Jwa et al. 1999).

We showed that Bfa1-D8391-574 bears the SPB-localizing and checkpoint signal-sensing activities of Bfa1, implying that the checkpoint activities of Bfa1p contained in D8391-574 likely depend on its association with Bub2p and localization to the SPB. This possibility was further investigated by isolating a point mutant of Bfa-D8, D8E438K, which loses the checkpoint functions of D8. Despite the lack of its checkpoint functions, D8E438K could localize to the SPB and interact with Bub2p and Tem1p. These results suggest that the localization of Bfa1p to the SPB by its binding to Bub2p is a prerequisite for sensing various checkpoint signals but not sufficient for checkpoint functions. In addition, our observation that the over-expression of D8E438K no longer induced mitotic arrest but could interact with Tem1p also suggest that the binding of Bfa1-D8 to Tem1p is not enough for negatively regulating Tem1p. The residue mutated in D8E438K was not the known phosphorylation site by Cdc5p. The new mutant we isolated demonstrates that the mutagenized glutamine residue is critical for the checkpoint functions of Bfa1p, possibly by directly regulating the GAP activity of Bfa1-D8 without affecting its interaction with Bub2p and Tem1p. We are currently investigating the GAP activity of Bfa1-D8E438K.

We showed that Bfa1-D8391-574 bears the SPB-localizing and checkpoint signal-sensing activities of Bfa1 although its localization to the SPB is not enough for checkpoint functions. These results still support that the localization of Bfa1p to the SPB and its binding to Bub2p are necessary for detecting the checkpoint signals. Then, why is the localization of Bfa1p to the SPB needed for sensing the defects to activate the checkpoint? Bfa1 is best known to prevent mitotic exit by recognizing the defects in spindle alignment. Two partially redundant pathways are involved in spindle orientation: the Kar9 and Bim1 pathway and a dynein-dependent pathway (Adames & Cooper 2000; Beach et al. 2000; Korinek et al. 2000; Farkasovsky & Kuntzel 2001). Recent reports showed that Kar9, Bim1, and dynein involved in the mitotic spindle orientation are present at the SPB as well as the microtubule-plus ends, suggesting that the SPB may play direct roles in spindle positioning for proper chromosome segregation (Segal & Bloom 2001; Liakopoulos et al. 2003; Maekawa et al. 2003). Considering that both Kar9 and Bfa1 are asymmetrically loaded on to the SPB that enters the bud (Li 1999; Liakopoulos et al. 2003; Maekawa et al. 2003), Bfa1p may be involved in sensing the defects in spindle misorientation to inhibit mitotic exit at the SPB. The N-terminal 376 amino acids of Bfa1p (D161-376) did not block mitotic exit in response to diverse checkpoint signals, while it did partially arrest cells in mitosis by over-expression and physically interact with Tem1p and Cdc5p. These observations demonstrate that various checkpoint signals cannot be transmitted to D16, although at least a part of mitotic arrest activity is conserved in D16. Also, D161-376 neither interacts with Bub2p nor localizes to the SPB, consistent with the previous report that the localization of Bfa1p and Bub2p to the SPB is interdependent (Pereira et al. 2000). As predicted from the lack of interaction between D16 and Bub2p, over-expression of D16 partially arrested cells in mitosis regardless of Bub2p, indicating that Bfa1p can regulate mitotic exit to some extent in the absence of Bub2p. These observations are consistent with the recent report showing that over-expression of Bfa1p efficiently arrests cell cycle at postanaphase in the absence of BUB2 and that Bfa1p can bind strongly with Tem1p independently of Bub2p (Ro et al. 2002). In addition, our data showed that mitotic arrest by Bfa1-D16 over-expression was relieved by concomitant over-expression of Tem1p. These observations strongly suggest that the mechanism that Bfa1p regulates Tem1p independent of Bub2p in the mitotic exit network as reported by Ro et al. (2002) is likely mediated by Bfa1-D16. Our data along with those of others suggest that Bfa1p acts not only in a two-component GAP with Bub2p at the SPB, it may also directly prevent Tem1p from binding with its effector(s) through D16, thereby inhibiting mitotic exit, when over-expressed. Over-expression of D16 was approximately 30% less efficient in preventing mitotic exit than D8 or full-length Bfa1p. Such lesser mitotic arrest by D16 could be ascribed to its lack of localization to the SPB and/or its poorer binding to Tem1p.

Hu et al. (2001) has shown that Bfa1p is inactivated by Cdc5p-dependent phosphorylations at 11 sites to trigger mitotic exit. The Bfa1 deletion mutants D161-376 contain nine of these phosphorylation sites while D8391-574 bears two. The mitotic exit defects of the CDC5 mutant alleles cdc5-1 and cdc5-2 are suppressed by deletion of BFA1 but were regained when D8391-574 was expressed. These two observations suggest that of the 11 Cdc5p-dependent phosphorylation sites, only the two sites mapped in D8391-574 are essential for the regulation of Bfa1p. However, when we examined a mutant D8391-574 protein containing point mutations at these two Cdc5p-dependent phosphorylation sites, we found that this protein also localized to SPB and arrested mitotic exit by over-expression (J. Kim and K. Song, unpublished observation). Thus, Bfa1p localization to the SPB is not affected by these mutations. The observation that a mutant D8391-574 protein containing point mutations at the residues phosphorylated by Cdc5p arrests mitotic exit when over-expressed is not surprising, however, since these phosphorylation sites are reported to negatively regulate Bfa1p. The loss of phosphorylation by mutations would block mitotic exit even if this protein were not over-expressed. Consistent with our results, a recent report showed that the phosphorylation of Bfa1p by Cdc5p reduces GAP activity with Bub2p (Geymonat et al. 2003).

In summary, we have shown that the major checkpoint functions of Bfa1p are concentrated in its C-terminal 184 residues and this C-terminal domain of Bfa1p is essential to respond to diverse checkpoint-activating signals. Given that our experiments did not reveal any functional discrepancies between the full-length Bfa1p and Bfa1-D8 in regulating mitotic checkpoint, the specific function of the N-terminal Bfa1p (Bfa1-D16) remains to be solved. Further studies on the interplay of Bfa1p with the proteins functioning in the spindle alignment, and the identification of the proteins that selectively interact with each Bfa1p domain will help to understand exactly how Bfa1p participates in the control of mitotic exit.


   Experimental procedures
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 Abstract
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 Results
 Discussion
 Experimental procedures
References
 
Yeast strains, cultures, cell cycle synchronization

The S. cerevisiae strains used in this study are listed in Table 1. Yeast cells were grown in YPDA medium (1% yeast extract, 2% bactopeptone, 2% glucose and 100 µg/mL adenine) or in synthetic complete (SC) drop-out media prepared with yeast nitrogen base (YNB) and the necessary supplements. To induce expression from the GAL10-1 promoter, cells grown to mid-log in 2% glucose were transferred to SC drop-out media with 2% raffinose for 14 h and then incubated in 2% raffinose/galactose for 4 h at 30 °C. Cells were synchronized by adding {alpha}-factor (Sigma) to a final concentration of 15 µg/mL or 0.2 M hydroxyurea (Sigma) for 3–4 h. The cells were released from cell cycle arrest by washing the cultures with fresh medium several times. Yeast strains were constructed using a PCR-based one-step gene disruption technique previously described (Longtine et al. 1998).


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  		Table 1  Yeast strains used in this study
 
Construction of BFA1 deletion mutants

DNA cloning and manipulation were performed using standard methods (Sambrook & Russell 2001). BFA1 deletion mutants were amplified by PCR using W303 chromosomal DNA as a template and the indicated oligonucleotides as primers (Table 2). PCR-amplified BFA1 deletion mutants were subcloned into pCEN-PGAL, pCEN-PGAL-GFP, pCEN-PBFA1 and pLexA202 +PL as described in Table 3. Each deletion mutant was inserted into pCEN-PGAL (a CEN-based vector with the GAL10-1 promoter) to observe the over-expression phenotypes. To investigate the localization of the Bfa1 deletion mutants, each mutant was subcloned into pCEN-PGAL-GFP (a pRS316-based C-terminal GFP-tagging vector with the GAL10-1 promoter). To assess the ability of Bfa1 deletion mutants to complement bfa1{Delta}, pCEN-PBFA1-BFA1 deletion mutants were constructed. The putative BFA1 promoter (about 500 bp upstream of open reading frame of BFA1) was PCR-amplified using oligonucleotides KS192 and KS193 and subcloned into pTS903CL (a CEN-based C-terminal 2 x HA and 6 x HIS epitope-tagging vector, a gift from A. Toh-e). Each PCR-amplified BFA1 deletion mutant was then inserted into pTS903CL-PBFA1. The pLexA202 +PL-BFA1 deletion mutants were constructed for two-hybrid assays. The putative BFA1 promoter (oligonucleotides KS192 and KS193) and BFA1-D8 or D8E438K (oligonucleotides KS162 and KS8) were amplified by PCR and subcloned into pRS306 to construct pRS306-PBFA1-BFA1-D8-3HA or D8E438K. pRS306-PBFA1-BFA1-D8-3HA or D8E438K were integrated into the chromosome of bfa1{Delta} (YSK8) to make strains YSK38 and YSK39. BFA1-D8-YFP SPC42-CFP bfa1{Delta} (YSK40) was constructed by the C-terminal genomic insertion of CFP into SPC42 using pDH3 (provided by Yeast Resource Center, University of Washington) and the integration of pRS304-PBFA1-BFA1-D8-YFP into bfa1{Delta} (YSK8).


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  		Table 2  Oligonucleotides used to construct the Bfa1 deletion mutants
 

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  		Table 3  Construction of the BFA1 deletion mutants
 
Microscopic techniques and fluorescence-activated cell sorter analysis

For fluorescence microscopy, cells were fixed in 70% ethanol or 3.7% formaldehyde, washed twice with PBS, briefly sonicated, and mounted with 1 µg/mL 4'6-diamidino-2-phenylindole (DAPI). Cells were observed with a 100X objective on an Axioplan2 (Zeiss) and images were captured with an Axiocam CCD (Zeiss) camera using AxioVision software (Zeiss). The DNA content of cells was examined by flow cytometry as described in Hwang & Song (2002). For each sample, 20 000 cells were analysed with a Becton Dickinson fluorescence-activated cell analyser.

Two-hybrid analyses

Two-hybrid assays were performed using a system described by Gyuris et al. (1993). BUB2, TEM1, and CDC5 were subcloned into pJG4-5 after amplification by PCR. The full-length and 13 deletion mutants of BFA1 were fused to the DNA-binding domain in pLexA202 +PL. The yeast strain EGY48 was co-transformed with these constructs and the reporter plasmid pSH18-34. Duplicate samples of each combination were subjected to X-gal overlay assays and quantitative ß-galactosidase assays as described by Jwa & Song (1998).

Protein techniques

To prepare cell lysates for immunoblots and co-precipitations, harvested cells grown to mid-log phase were extracted in modified H-buffer [25 mM Tris-HCl (pH 7.4), 150 mM NaCl, 15 mM MgCl2, 15 mM EGTA (pH 7.5), 0.1% Triton X-100, 10% glycerol, 1 mM NaN3, 1 mM DTT, 0.5 mM phenylmethylsulphonyl fluoride (PMSF), protease inhibitor cocktail (Boeringer Mannheim)] by beadbeating (Biospec) and clarified by 14 000 r.p.m. centrifugation at 4 °C. For co-precipitation assays of Bfa1 deletion mutants with Tem1p or Bub2, GFP-tagged Bfa1 deletion mutants, and 3 x HA-tagged Tem1p and Bub2p under GAL10-1 promoter were, respectively, introduced into bfa1{Delta} (YSK8), separately expressed, and verified by immunoblots. Each lysate of separately expressed Bfa1 deletion mutants tagged with GFP and Tem1p or Bub2p tagged with 3 x HA was mixed together and purified with anti-HA antibody followed by protein A-agarose (Sigma). Co-precipitates were blotted with affinity-purified polyclonal anti-GFP (Santa Cruz) and monoclonal anti-HA (Roche), as described essentially by Hwang & Song (2002). For co-precipitation assays of Bfa1-D8E438K with Bub2p or Tem1p, 3xHA-tagged D8E438K and GST-tagged Bub2p and Tem1p under GAL10-1 promoter were, respectively, introduced into bfa1{Delta} (YSK8), separately expressed, and verified by immunoblots. Each lysate was mixed, purified with glutathione-agarose (Sigma), and co-precipitates were blotted with anti-GST (Upstate) and anti-HA (Roche) antibodies.

Random mutagenesis of plasmid DNA

Random in vitro mutagenesis was performed with hydroxylamine. Approximately 10 µg of plasmid pRS316-PBFA1-BFA1-D8 was incubated in 0.5 mL hydroxylamine solution (1 M hydroxylamine, 50 mM sodium pyrophosphate (pH 7.0), 100 mM sodium chloride, 2 mM EDTA) at 75 °C for 90 min. After the mixture had been cooled on ice, DNA was separated by gel filtration (Sephadex G-25) and the mutagenized plasmid pRS316-PBFA1-BFA1-D8 was introduced into bfa1{Delta} yeast strain (YSK8). Transformants were replica plated and screened for the loss of viability in the presence of 10 µg/mL benomyl. Screened mutant plasmids were restored, sequenced, and re-subcloned into the intact pRS306-PBFA1-3HA for verifying the sensitivity to benomyl.


   Acknowledgements
 
We thank Drs Kelly Tatchell, John Chant, Akio Toh-e, Takeshi Sasaki, Martine Lonetine, John Cooper, David Pellman, Stephen Elledge, and Kyung S. Lee for their generous gifts of yeast strains and plasmids. This work was supported by grants from the Korean Ministry of Science and Technology (M10309000002-03B5000-00110) and a grant (ROI-2003-000-1049800) from KOSEF on ‘Systems Biology’ donated to K. Song. J. Kim and J. Jeong were supported by the Brain Korea 21 Project of 2003.


   Footnotes
 
Communicated by: Masayuki Yamamoto

* Correspondence: E-mail: bc5012{at}yonsei.ac.kr


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Adames, N.R. & Cooper, J.A. (2000) Microtubule interactions with the cell cortex causing nuclear movements in Saccharomyces cerevisiae. J. Cell Biol. 149, 863–874.[Abstract/Free Full Text]

Adames, N.R., Oberle, J.R. & Cooper, J.A. (2001) The surveillance mechanism of the spindle position checkpoint in yeast. J. Cell Biol. 153, 159–168.[Abstract/Free Full Text]

Asakawa, K., Yoshida, S., Otake, F. & Toh-e, A. (2001) A novel functional domain of Cdc15 kinase is required for its interaction with Tem1 GTPase in Saccharomyces cerevisiae. Genetics 157, 1437–1450.[Abstract/Free Full Text]

Bardin, A.J., Visintin, R. & Amon, A. (2000) A mechanism for coupling exit from mitosis to partitioning of the nucleus. Cell 102, 21–31.[CrossRef][Medline]

Beach, D.L., Thibodeaux, J., Maddox, P., Yeh, E. & Bloom, K. (2000) The role of the proteins Kar9 and Myo2 in orienting the mitotic spindle of budding yeast. Curr. Biol. 10, 1497–1506.[CrossRef][Medline]

Bloecher, A., Venturi, G.M. & Tatchell, K. (2000) Anaphase spindle position is monitored by the BUB2 checkpoint. Nature Cell Biol. 2, 556–558.[CrossRef][Medline]

Farkasovsky, M. & Kuntzel, H. (2001) Cortical Num1p interacts with the dynein intermediate chain Pac1p and cytoplasmic microtubules in budding yeast. J. Cell Biol. 152, 251–262.[Abstract/Free Full Text]

Furge, K.A., Cheng, Q., Jwa, M., Shin, S., Song, K. & Albright, C.F. (1999) Regions of Byr4, a regulator of septation in fission yeast, that bind Spg1 or Cdc16 and form a two-component GTPase-activating protein with Cdc16. J. Biol. Chem. 274, 11339–11343.[Abstract/Free Full Text]

Furge, K.A., Wong, K., Armstrong, J., Balasubramanian, M. & Albright, C.F. (1998) Byr4 and Cdc16 form a two-component GTPase-activating protein for the Spg1 GTPase that controls septation in fission yeast. Curr. Biol. 8, 947–954.[CrossRef][Medline]

Garvik, B., Carson, M. & Hartwell, L. (1995) Single-stranded DNA arising at telomeres in cdc13 mutants may constitute a specific signal for the RAD9 checkpoint. Mol. Cell. Biol. 15, 6128–6138.[Abstract]

Geymonat, M., Spanos, A., Smith, S.J., et al. (2002) Control of mitotic exit in budding yeast: In vitro regulation of Tem1 GTPase by Bub2 and Bfa1. J. Biol. Chem. 277, 28439–28445.[Abstract/Free Full Text]

Geymonat, M., Spanos, A., Walker, P.A., Johnston, L.H. & Sedgwick, S.G. (2003) In vitro regulation of budding yeast Bfa1/Bub2 GAP activity by Cdc5. J. Biol. Chem. 278, 14591–14594.[Abstract/Free Full Text]

Gyuris, J., Golemis, E., Chertkov, H. & Bren, R. (1993) Cdi1, a human G1 and S phase protein phosphatase that associates with Cdk2. Cell 75, 791–803.[CrossRef][Medline]

Hoyt, M.A. (2000) Exit from mitosis: spindle pole power. Cell 102, 267–270.[CrossRef][Medline]

Hu, F., Wang, Y., Liu, D., Li, Y., Qin, J. & Elledge, S.J. (2001) Regulation of the Bub2/Bfa1 GAP complex by Cdc5 and cell cycle checkpoints. Cell 107, 655–665.[CrossRef][Medline]

Hwang, H.S. & Song, K. (2002) IBD2 encodes a novel component of the Bub2p-dependent spindle checkpoint in the budding yeast Saccharomyces cerevisiae. Genetics 161, 595–609.[Abstract/Free Full Text]

Janke, C., Ortiz, J., Tanaka, T.U., Lechne, J. & Schiebel, E. (2002) Four new subunits of the Dam1-Duo1 complex reveal novel functions in sister kinetochore biorientation. EMBO J. 21, 181–193.[CrossRef][Medline]

Jaspersen, S.L., Charles, J.F. & Morga, D.O. (1999) Inhibitory phosphorylation of the APC regulator Hct1 is controlled by the kinase Cdc28 and the phosphatase Cdc14. Curr. Biol. 9, 227–236.[CrossRef][Medline]

Jwa, M., Shin, S., Albright, C.F. & Song, K. (1999) Byr4p, a possible regulator of mitosis and cytokinesis in fission yeast, localizes to the spindle pole body by its C-terminal domains. J. Biochem. Mol. Biol. 32, 92–97.

Jwa, M. & Song, K. (1998) Byr4, a dosage-dependent regulator of cytokinesis in S. pombe, interacts with a possible small GTPase pathway including Spg1 and Cdc16. Mol. Cells 8, 240–245.[Medline]

Knapp, D., Bhoite, L., Stillman, D.J. & Nasmyth, K. (1996) The transcription factor Swi5 regulates expression of the cyclin kinase inhibitor p40Sic1. Mol. Cell. Biol. 16, 5701–5707.[Abstract]

Korinek, W.S., Copeland, M.J., Chaudhuri, A. & Chan, J. (2000) Molecular linkage underlying microtubule orientation toward cortical sites in yeast. Science 287, 2257–2259.[Abstract/Free Full Text]

Lee, S.E., Frenz, L.M., Wells, N.J., Johnso, A.L. & Johnston, L.H. (2001) Order of function of the budding-yeast mitotic exit-network proteins Tem1, Cdc15, Mob1, Dbf2, and Cdc5. Curr. Biol. 11, 784–788.[CrossRef][Medline]

Lee, J., Hwang, H.S., Kim, J. & Song, K. (1999) Ibd1p, a possible spindle pole body associated protein, regulates nuclear division and bud separation in Saccharomyces cerevisiae. Biochim. Biophys. Acta 1449, 239–253.[Medline]

Li, R. (1999) Bifurcation of the mitotic checkpoint pathway in budding yeast. Proc. Natl. Acad. Sci. USA 96, 4989–4894.[Abstract/Free Full Text]

Li, Y., Bachant, J., Alcasabas, A.A., Wang, Y., Qin, J. & Elledge, S.J. (2002) The mitotic spindle is required for loading of the DASH complex onto the kinetochore. Genes Dev. 16, 183–197.[Abstract/Free Full Text]

Liakopoulos, D., Kusch, J., Grava, S., Vogel, J. & Barral, Y. (2003) Asymmetric loading of Kar9 onto spindle poles and microtubules ensures proper spindle alignment. Cell 112, 561–574.[CrossRef][Medline]

Longtine, M.S., McKenzie, A., 3rd, Demarini, D.J. , et al. (1998) Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14, 953–961.[CrossRef][Medline]

Maekawa, H., Usui, T., Knop, M. & Schiebel, E. (2003) Yeast Cdk1 translocates to the plus end of cytoplasmic microtubules to regulate bud cortex interactions. EMBO J. 22, 438–449.[CrossRef][Medline]

McCollum, D. & Gould, K.L. (2001) Timing is everything: regulation of mitotic exit and cytokinesis by the MEN and SIN. Trends Cell Biol. 11, 89–95.[CrossRef][Medline]

Morgan, D.O. (1999) Regulation of the APC and the exit from mitosis. Nature Cell Biol. 1, E47–E53.[CrossRef][Medline]

Pereira, G., Hofken, T., Grindlay, J., Manson, C. & Schiebel, E. (2000) The Bub2p spindle checkpoint links nuclear migration with mitotic exit. Mol. Cell 6, 1–10.[CrossRef][Medline]

Pereira, G., Tanaka, T.U., Nasmyth, K. & Schiebel, E. (2001) Modes of spindle pole body inheritance and segregation of the Bfa1p-Bub2p checkpoint protein complex. EMBO J. 20, 6359–6370.[CrossRef][Medline]

Ro, H.S., Song, S. & Le, K.S. (2002) Bfa1 can regulate Tem1 function independently of Bub2 in the mitotic exit network of Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 99, 5436–5441.[Abstract/Free Full Text]

Sambrook, J. & Russell, D.W. (2001) Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor, N.Y: Cold Spring. Harbor Laboratory Press.

Schwab, M., Lutum, A.S. & Seufert, W. (1997) Yeast Hct1 is a regulator of Clb2 cyclin proteolysis. Cell 90, 683–693.[CrossRef][Medline]

Segal, M. & Bloom, K. (2001) Control of spindle polarity and orientation in Saccharomyces cerevisiae. Trends Cell Biol. 11, 160–166.[CrossRef][Medline]

Shou, W., Seol, J.H., Shevchenko, A., et al. (1999) Exit from mitosis is triggered by Tem1-dependent release of the protein phosphatase Cdc14 from nucleolar RENT complex. Cell 97, 233–244.[CrossRef][Medline]

Song, K., Mach, K.E., Chen, C.Y., Reynold, T. & Albright, C.F. (1996) A novel suppressor of ras1 in fission yeast, byr4, is a dosage-dependent inhibitor of cytokinesis. J. Cell Biol. 133, 1307–1319.[Abstract/Free Full Text]

Visintin, R., Craig, K., Hwang, E.S., Prinz, S., Tyers, M. & Amon, A. (1998) The phosphatase Cdc14 triggers mitotic exit by reversal of Cdk-dependent phosphorylation. Mol. Cell 2, 709–718.[CrossRef][Medline]

Visintin, R., Hwang, E.S. & Amon, A. (1999) Cfi1 prevents premature exit from mitosis by anchoring Cdc14 phosphatase in the nucleolus. Nature 398, 818–823.[CrossRef][Medline]

Wang, Y., Hu, F. & Elledge, S.J. (2000) The Bfa1/Bub2 GAP complex comprises a universal checkpoint required to prevent mitotic exit. Curr. Biol. 10, 1379–1382.[CrossRef][Medline]

Zachariae, W., Schwab, M., Nasmyth, K. & Seufert, W. (1998) Control of cyclin ubiquitination by CDK-regulated binding of Hct1 to the anaphase promoting complex. Science 282, 1721–1724.[Abstract/Free Full Text]

Received: 3 September 2003
Accepted: 3 February 2004




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J. Kim, S. S. Jang, and K. Song
Different Levels of Bfa1/Bub2 GAP Activity Are Required to Prevent Mitotic Exit of Budding Yeast Depending on the Type of Perturbations
Mol. Biol. Cell, October 1, 2008; 19(10): 4328 - 4340.
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