Article |
Address correspondence to Elmar Schiebel, The Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Rd., Manchester, M20 4BX, UK. Tel.: 44-161-446-3783. Fax: 44-161-446-3109. email: eschiebel{at}picr.man.ac.uk
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: cell polarity; Cdc14; Gic proteins; Lte1; MEN
Abbreviations used in this paper: APC/C, anaphase-promoting complex; CRIB, Cdc42/Rac interactive binding; Cys, cysteine; FEAR, cdc fourteen early anaphase release; GAP, GTPase-activating protein; MBP, maltose binding protein; MEN, mitotic exit network; SPB, spindle pole body.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The small Ras-like GTPase Tem1 is one of the most upstream components of the MEN (Mah et al., 2001). The two-component GTPase-activating protein (GAP) Bfa1Bub2 complex keeps Tem1 in the inactive GDP-bound form (Shirayama et al., 1994a; Geymonat et al., 2002). Tem1, Bfa1, and Bub2 preferentially localize to the yeast centrosome, the spindle pole body (SPB), that is destined to enter the bud in anaphase. The SPB that stays in the mother cell does not carry Bfa1, Bub2, or Tem1 (Fraschini et al., 1999; Pereira et al., 2001). The MEN activator Lte1 that functions upstream of Tem1 and shows homology to the Ras guanine nucleotide exchange factor protein Cdc25 associates in a polar fashion with the cell cortex of small- and medium-sized buds. This association of Lte1 is lost mid way through anaphase (Bardin et al., 2000; Pereira et al., 2000; Seshan et al., 2002; Yoshida et al., 2003). As a consequence of the polar cellular distribution of Bub2-Bfa1, Lte1, and Tem1, MEN activation only occurs after the anaphase spindle has extended into the bud (Bardin et al., 2000; Pereira et al., 2000). Mitotic exit and cytokinesis thus become dependent on the successful elongation of the anaphase spindle into the bud.
Although most MEN components are essential, the deletion of the MEN activator LTE1 has no obvious phenotype at temperatures between 30°C and 37°C (Shirayama et al., 1994a; Adames et al., 2001; Pereira et al., 2002) suggesting that alternative pathways control mitotic exit. For example, polo-like kinase Cdc5 activates the MEN through inhibitory phosphorylation of the Tem1 GAP component Bfa1 (Hu et al., 2001). In addition, the Rho-like GTPase Cdc42, a key regulator of polarized growth, activates mitotic exit by at least two mechanisms through the Cdc42 effectors Ste20 and Cla4, two p21-activated kinases (Höfken and Schiebel, 2002; Jensen et al., 2002; Seshan et al., 2002). Cla4 targets Lte1 to the bud cortex and regulates both activity and phosphorylation of Lte1 (Höfken and Schiebel, 2002; Seshan et al., 2002). In contrast, Ste20 facilitates mitotic exit in a pathway that is redundant with LTE1 (Höfken and Schiebel, 2002).
Here we show that the Cdc42 effectors Gic1 and Gic2 can promote mitotic exit. Gic proteins become essential for mitotic exit when MEN activation through Cdc5 polo kinase and Lte1 are impaired. Our data are consistent with a model in which Gic1 binds to Bub2 and prevents the assembly of the Bub2Tem1 complex. Release of Gic1 from the bud cortex in anaphase is important for this regulation. This may provide an additional mechanism by which the elongation of the anaphase spindle into the bud is coordinated with the activation of mitotic exit.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
TEM1 was identified as a class one suppressor (Table I) as it functions downstream of LTE1 in the MEN (Shirayama et al., 1994b). SIC1 was a class two suppressor because it promotes mitotic exit by inhibiting Cdc28-Clb2 (Schwob et al., 1994). SPO12 and PUP3 also allowed growth of lte1
ste20 cells. SPO12 is a component of the FEAR network that is frequently obtained in genetic screens for mitotic exit components (Jaspersen et al., 1998; Stegmeier et al., 2002). PUP3 encodes a proteasome subunit (Finley et al., 1998). The mechanism by which PUP3 restored viability to
lte1
ste20 cells requires further studies. The most frequently isolated suppressor was BEM1 (Table I; Fig. 1 A). Bem1 acts as a scaffold protein that links Cdc42 with its guanine nucleotide exchange factor Cdc24 and downstream effectors such as Ste20 and Cla4. Bem1 stabilizes, as part of a positive feedback loop, active Cdc24 at sites of polarized growth and thus ensures continuous production of active Cdc42 (Bose et al., 2001; Butty et al., 2002). Therefore, it was not surprising that high gene dosage of CDC42 and of the Cdc42 activator CDC24 also suppressed
lte1
ste20 lethality (Table I, Fig. 1 A).
|
|
Taking the previously established interaction of Cdc24, Cdc42, and Gic1 (Brown et al., 1997; Chen et al., 1997; Butty et al., 2002) into consideration with our suppression data, it seemed likely that we had identified a pathway in which a signal is transmitted from Cdc24 to Cdc42 and hence to the Cdc42 effectors Gic1 and Gic2. The latter two would then promote mitotic exit.
Next, we investigated how GIC1 and GIC2 facilitate mitotic exit. Gic proteins could directly activate the APC/C specificity factor Hct1/Cdh1, the Wee1-like kinase Swe1 or the Cdk1 inhibitor Sic1 (Booher et al., 1993; Schwob et al., 1994; Schwab et al., 1997). However, 2µm-GIC1 allowed growth of lte1
ste20 cells with similar efficiency whether HCT1/CDH1 or SWE1 function was present or not (unpublished data). In addition, the growth defect of
sic1 and
lte1
sic1 cells, which are delayed in mitotic exit (not depicted), was rescued by GIC1 and GIC2 to the same extent as by TEM1 (Fig. 1 D). This suggests that GIC1 and GIC2 promote mitotic exit independently of HCT1/CDH1, SIC1, and SWE1.
GIC genes could trigger the release of Cdc14 from the nucleolus. This possibility was addressed by turning to the well-characterized lte1 cells, which arrest in late anaphase due to an inability to activate the MEN (Shirayama et al., 1994b; Höfken and Schiebel, 2002). The cold sensitive growth defect of
lte1 cells was suppressed by high gene dosage of BEM1, CDC24, CDC42, GIC1, and GIC2 (Fig. 1 E, 10°C). Then, we asked whether the failure of
lte1 cells at 14°C to release Cdc14 from the nucleolus was rescued by GIC1 and GIC2. Cells were synchronized in G1 and allowed to progress into a new cell cycle at 14°C. Bud growth and release of Cdc14-GFP from the nucleolus were monitored as the synchronized populations divided. Consistent with published data (Shou et al., 1999; Visintin et al., 1999), Cdc14 was released from the nucleolus of wild-type cells as they entered anaphase (Fig. 1 F, t = 6 h) and was taken up by the nucleolus as cell exited mitosis (Fig. 1 F, t = 810 h). In contrast, in virtually all of the
lte1 cells Cdc14 remained bound in the nucleolus and
90% of cells arrested as large budded, binucleated cells in late anaphase (Fig. 1 F).
lte1 cells overexpressing GIC1 or GIC2 released Cdc14 from the nucleolus with similar kinetics and efficiency as wild-type cells. The decrease of cells with a large bud and the reuptake of Cdc14 into the nucleolus indicated that these cells successfully exited mitosis (Fig. 1 F). Thus, GIC1 and GIC2 suppressed the mitotic exit defect of
lte1 cells by facilitating the release of Cdc14 from the nucleolus. This in turn implies that the Gic proteins activate either the FEAR or MEN networks which control Cdc14.
The mitotic exit function of Gic proteins becomes essential when Cdc5 and Lte1 are impaired
GIC1 and GIC2 promote mitotic exit by facilitating the release of Cdc14 from the nucleolus. Therefore, cells lacking GIC genes should, at least under certain conditions, have a mitotic exit defect. Single or double deletion of GIC genes did not reveal any obvious mitotic exit delay at 30°C (Fig. 2 C). At 37°C, gic1
gic2 cells arrest as unbudded cells due to a defect in actin polarization (Brown et al., 1997; Chen et al., 1997). One possible interpretation of this observation is that several mechanisms promote mitotic exit and due to this redundancy the function of Gic proteins in mitotic exit only becomes apparent when the other pathways are also impaired.
|
To analyze the cell cycle phenotype of cdc5-10 gic1
gic2
lte1 cells, we generated conditional lethal cells by placing the BUB2 gene under control of the Gal1 promoter. cdc5-10
gic1
gic2
lte1 Gal1-BUB2 cells grown at 30°C in the presence of glucose (repression of Gal1-BUB2) were viable (Fig. 2 B) and progressed through the cell cycle similarly to wild-type cells (not depicted). Addition of galactose to induce expression of Gal1-BUB2 restored BUB2 function and the lethal phenotype of cdc5-10
gic1
gic2
lte1 cells (Fig. 2 B). We were now able to analyze the phenotype of cdc5-10
gic1
gic2
lte1 cells.
-Factor synchronized cells carrying CDC14-GFP and Gal1-BUB2 (Fig. 2 C) were released into a new cell cycle after the addition of galactose (t = 0) to induce BUB2 expression. Expression of BUB2 from the Gal1 promoter did not affect cell cycle progression of wild-type, cdc5-10, or cdc5-10
gic1
gic2 cells (not depicted) nor did it delay mitotic exit in these cells, which would become apparent through a plateau of cells with a large bud (Fig. 2 C). Remarkably, at 30°C, wild-type, cdc5-10
lte1, and
gic1
gic2 cells released Cdc14-GFP from the nucleolus with similar kinetics and efficiency (maximum at
120 min) and then exited mitosis as indicated by the decrease of large-budded cells, the reuptake of Cdc14 into the nucleolus (Fig. 2 C), and the degradation of the mitotic cyclin Clb2 (Fig. 2 D). Moreover, mitotic exit was accompanied by the accumulation of the Cdk1-Clb inhibitor Sic1 in wild-type and, to a somewhat lesser extent, in cdc5-10
lte1 and
gic1
gic2 cells (Fig. 2 D). In contrast,
90% cdc5-10
gic1
gic2
lte1 cells arrested in anaphase with a large bud, separated DAPI staining regions, a long anaphase spindle, Cdc14-GFP trapped in the nucleolus (Fig. 2, C and E), high Clb2 levels and no accumulation of Sic1 (Fig. 2 D). This combination of phenotypes is the hallmark of cells with a defect in mitotic exit (Pereira and Schiebel, 2001). Thus, cdc5-10
gic1
gic2
lte1 cells execute anaphase at 30°C similarly to wild-type cells but then fail to exit mitosis.
Gic1 disrupts the formation of the Bfa1Bub2Tem1 complex
We used the yeast two-hybrid system to test whether Gic1 interacted with proteins involved in mitotic exit. Strong two-hybrid interactions were detected between Gic1 and Cdc42 (Brown et al., 1997; Chen et al., 1997), Bfa1 and Cdc14 (Fig. 3 A). Consistent with published results (Uetz et al., 2000; Drees et al., 2001), we also observed a two-hybrid interaction between Gic1 and Bub2 (Fig. 3 A). In contrast, Gic2 constructs failed to show two-hybrid interactions even with Cdc42 (unpublished data). Thus, fusion to the Gal4 and LexA elements of the two-hybrid system probably impaired the activity of GIC2.
|
Gic1 could activate mitotic exit by releasing Cdc14 from the inhibitory Net1Cdc14 complex, however, two results suggest that this is unlikely. First, overexpression of GIC1 from the strong Gal1 promoter did not release Cdc14 from the nucleolus of cells arrested in metaphase by depletion of the APC/C subunit Cdc20 (Fig. S1 A, available at http://www.jcb.org/cgi/content/full/jcb.200309080/DC1). Second, when a preformed Cdc14Net1 complex was incubated in vitro with increasing amounts of purified Gic1 protein, Cdc14 was not displaced from Net1 (Fig. S1 B). In contrast, increasing amounts of recombinant Net1 displaced Cdc14 from Gic1. Together, these results suggest that Gic1 does not promote mitotic exit by directly regulating Cdc14 localization. The functional relevance of the Cdc14Gic1 interaction remains unclear.
A second possible mechanism by which Gic1 could regulate mitotic exit is by interfering with the formation of the inhibitory Bfa1Bub2Tem1 complex. We investigated whether Gic1 prevented the interaction of Tem1 with either Bub2 or Bfa1. Purified His6-Tem1 was preincubated with increasing amounts of GST-Gic1 or GST. The proteins were then incubated with purified, immobilized MBP, MBP-Bub2 or MBP-Bfa1. Tem1 and Gic1 failed to bind to MBP beads (unpublished data). Binding of Tem1 to Bub2 and Bfa1 was observed in the absence of Gic1 (Fig. 4, A and B, lane 1). The addition of GST-Gic1 (Fig. 4, A and B, lanes 26) but not GST (lanes 812) decreased His6-Tem1 binding to both Bub2 (Fig. 4 A, top) and Bfa1 (Fig. 4 B, top). It is important to note that Gic1 was 10 times more efficient in inhibiting Tem1 binding to Bub2 than inhibiting its association with Bfa1 (Fig. 4, A and B, compare lanes 14). Therefore, in vitro Gic1 preferentially inhibits Tem1 binding to Bub2.
|
Gic1-induced disruption of complex formation between Bub2 and Tem1 was also observed in vivo. Coimmunoprecipitation of Bub2 and Tem1 from yeast cell extracts has been reported previously (Pereira et al., 2000). We asked whether elevated Gic1 levels reduced the efficiency of this coimmunoprecipitation. In the presence of the pRS425 control plasmid the anti-Myc antibodies not only efficiently immunoprecipitated Tem1-9Myc but also Bub2-3HA (Fig. 4 D, lane 8). This coimmunoprecipitation was dependent on the presence of Myc-tagged Tem1 (Fig. 4 D, lane 7) indicating specificity of the precipitation. High gene dosage of GIC1 but not of the inactive 2µm-GIC1CRIB- strongly reduced the efficiency of the coimmunoprecipitation of Tem1-9Myc and Bub2-3HA (Fig. 4 D, lanes 9 and 10). In contrast, 2µm-GIC1 did not affect the ability to coprecipitate Bfa1-Tem1 and Bfa1-Bub2 (unpublished data). This selectivity is consistent with our in vitro data indicating preferential inhibition of Tem1Bub2 interaction by Gic1 (Fig. 4, AC). Thus, Gic1 inhibits binding of Tem1 to Bub2 in vivo and in vitro.
Gic1 is a stable protein whose localization changes during the cell cycle
Gic2 has been reported to be subject to cell cycledependent degradation. In particular, Gic2 is absent from G2 cells (Brown et al., 1997). This raises the question as to how Gic1 and Gic2 can regulate mitotic exit when they are not present in mitosis. Reevaluation of Gic levels during the cell cycle revealed that Gic2 was partially degraded with bud emergence (Fig. 5 B, t = 70 min) but a considerable fraction persisted even after cells had entered mitosis. Gic1 protein levels did not fluctuate during the cell cycle (Fig. 5 A). The appearance of multiple bands in the SDS-PAGE indicated that Gic1 was subject to cell cycledependent modification in G1 when cells were predominantly unbudded (Fig. 5 A, t = 50 min). Thus, both Gic proteins are present in mitosis.
|
The Gic1 which is associated with the bud cortex organizes the actin cytoskeleton but does not promote mitotic exit
The distribution of Gic1 suggests two possible mechanisms by which it may regulate mitotic exit. The bud cortex associated Gic1 could disrupt the inhibitory Bfa1Bub2Tem1 complex when the SPB carrying these proteins streaks along the bud cortex in anaphase (Adames and Cooper, 2000). Alternatively, Gic1 released from the bud cortex could prevent Tem1Bub2 complex formation. To investigate whether the membrane associated Gic1 is sufficient to promote mitotic exit, we permanently anchor Gic1 to the plasma membrane. The COOH terminus of Ras2 (aa 301 to 322) can target a protein to the plasma membrane (Fig. 6 A, Gic1-pr; Pryciak and Huntress, 1998). This is because cysteine (Cys) 318 of Ras2 becomes palmitylated and Cys 319 becomes prenylated (Mitchell et al., 1994). To control for the possibility that the fusion of this sequence per se affects Gic1 function, Cys 318 and 319 of the Ras2 element were mutated to serine (Fig. 6 A, Gic1-pr-SS). These mutations prevent modification of Ras2301-322 and the permanent anchorage of Gic1-pr-SS to the plasma membrane.
|
The functionality of GIC1-pr and GIC1-pr-SS was tested. gic1
gic2 cells are unable to grow at 37°C because of a defect in actin polarization (Brown et al., 1997; Chen et al., 1997). We introduced wild-type and mutant GIC1 genes on the CEN-based low copy number vector pRS315 into
gic1
gic2 cells. GIC1, GIC1-pr, and GIC1-pr-SS were equally efficient in complementing the growth defect of
gic1
gic2 cells at 37°C (Fig. 6 C). Staining F-actin with rhodamine-phalloidin showed that most
gic1
gic2 pRS315 cells accumulated as enlarged unbudded cells, which lacked a polarized actin cytoskeleton (Fig. 6 D). pRS315-GIC1-pr restored the actin cytoskeleton (Fig. 6 D) to the same degree as seen for GIC1 or GIC1-pr-SS (not depicted). Thus, the membrane bound Gic1-pr was able to fulfil the actin polarization function of Gic proteins.
Next, we investigated whether the membrane bound Gic1-pr suppressed the mitotic exit defect of lte1
ste20 cells. Although GIC1-pr-SS on a 2µm high copy plasmid was as potent as GIC1 in suppressing the growth defect of
lte1
ste20 cells, GIC1-pr failed to support growth (Fig. 6 E). Consistently, only GIC1-pr-SS but not the membrane anchored GIC1-pr was able to suppress the mitotic exit defect of synchronized
lte1 cells at 14°C (Fig. 1 F). Moreover, the lack of Gic1-pr to promote mitotic exit correlated with the incapability of this protein to disrupt the Bub2Tem1 interaction in vivo (Fig. 4 D, lane 11). In contrast, the mitotic exit promoting GIC1-pr-SS interfered with Bub2Tem1 interaction (Fig. 4 D, lane 12). The failure of Gic1-pr to stimulate mitotic exit and to interfere with Bub2Tem1 interaction in vivo was not because the level of the Gic1-pr was preferentially decreased (Fig. 6 F) or because the pr-fusion enabled Gic1 to disrupt the Bub2Tem1 interaction in vitro (Fig. 6 G). The latter result rather suggests that the reason why Gic1-pr did not reduce the Bub2Tem1 interaction was the permanent association with the bud cortex. Thus, the membrane bound Gic1 is unable to promote mitotic exit and to disrupt the Bub2Tem1 interaction.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Gic proteins triggered the nucleolar release of Cdc14 in lte1 cells at 14°C. Thus, Gic proteins activate either the MEN or the FEAR network, the two known pathways that regulate Cdc14 (Stegmeier et al., 2002), or directly regulate Cdc14. Gic1 interacted with Bfa1, Bub2, Tem1, and Cdc14 by two-hybrid and in vitro binding studies. Attempts to show these interactions by coimmunoprecipitation failed, however, this is likely because of the very transient nature of the interactions. Other established interactions of MEN components such as binding of Cdc14 to Bfa1 and of Cdc15 to Dbf2 cannot be seen by coimmunoprecipitation assays (Pereira et al., 2002; Visintin et al., 2003). Gic1 could disrupt the nucleolar Net1Cdc14 complex and by this means release Cdc14 from the nucleus. However, the failure of Gic1 to release Cdc14 from Net1 in vivo and in vitro argues against this possibility. Instead, our in vitro and in vivo data suggest that Gic1 activates mitotic exit by inhibiting the interaction between Tem1 and Bub2. Disruption of the Bub2Tem1 complex correlated with the capability of Gic1 to promote mitotic exit (Fig. 4 D). In contrast, a Gic1 protein lacking the activating CRIB domain failed both to promote mitotic exit and to disrupt the Bub2Tem1 complex. A similar behavior was observed for the membrane anchored Gic1-pr. A function upstream or in parallel of TEM1 is also supported by the observation that GIC1 does not suppress the mitotic exit and Cdc14-release defect of tem1-3 cells (unpublished data).
The membrane bound, cytoplasmic, and nuclear forms of Gic1 could promote mitotic exit. The finding that a Gic1 protein permanently anchored to the plasma membrane was unable to promote mitotic exit (Fig. 1 F and Fig. 6 E) suggests that Gic1 has to be released from the bud cortex after its activation by Cdc42 in order to promote mitotic exit. In light of this dependency, we propose that upon anaphase onset Gic1 becomes activated by Cdc42 at the bud cortex and is then released into the cytoplasm. The soluble Gic1 then contributes to the inactivation of the Bub2-Bfa1 GAP. The half-life of Cdc42-activated Gic1 may be short after its release from the bud cortex. This would ensure that Gic1 only inactivates the Bub2Bfa1Tem1 complex in the bud. Such a mechanism could contribute to the coupling of anaphase spindle elongation into the bud and mitotic exit.
The observation that Gic1-pr polarizes the actin cytoskeleton but fails to promote mitotic exit demonstrates that Gic1 has two separate functions. The established role of Gic1 in bud formation and actin polarization (Brown et al., 1997; Chen et al., 1997) at the bud cortex is distinct from the cell cycle promoting function that requires soluble Gic1 (this paper). However, Cdc42 regulates both functions because they depend on the Cdc42-binding CRIB domain within Gic1 (Brown et al., 1997; Chen et al., 1997).
The question remains as to where the regulation of the Bub2-Bfa1 GAP and the GTPase Tem1 takes place? The Bub2Bfa1Tem1 complex is enriched at the SPB (Fraschini et al., 1999; Pereira et al., 2000) but the importance of this localization is unclear (Pereira and Schiebel, 2001). For example, Tem1 also was found in complexes with the bud cortexassociated kelch domain proteins Kel1 and Kel2 (Höfken and Schiebel, 2002). This Tem1 pool and not the SPB associated Tem1 may be important for mitotic exit. In this respect it is interesting that the Tem1 regulators Lte1 and Amn1 are also not enriched at SPBs (Pereira et al., 2000; Wang et al., 2003). Thus, it is possible that Gic1 activates Tem1 by disrupting the function of a soluble Bub2Tem1 complex. Alternatively, the SPB associated Bub2-Bfa1-Tem1 could be the target of Gic1. Because of the high levels of cytoplasmic and nuclear Gic1, it is difficult to evaluate whether Gic1 is enriched at SPBs. In any case, it is likely that the cytoplasmic Gic1 at least transiently interacts with the Bub2Bfa1Tem1 complex at SPBs. This interaction could result in the displacement of Tem1 from SPBs, or simply inhibit Bub2-Bfa1 GAP activity without affecting Tem1 SPB binding. The finding that 2µm-GIC1 does not affect the Tem1-GFP fluorescence signal at SPBs (unpublished data) would favor the second possibility.
We propose that up until metaphase the MEN remains inactive because of the lack of Bfa1 phosphorylation and the binding of both Lte1 and Gic1 to the bud cortex. Upon anaphase onset, Cdc5, Gic1, and Lte1 concertedly activate the MEN (Fig. 7). The role of Lte1 in MEN activation is not fully understood. In fact, a recent report questioned a direct activation of Tem1 by Lte1 (Yoshida et al., 2003). Suppression of the lte1 mitotic exit defect by 2µm-GIC1 either means that GIC1 functions downstream or in parallel with LTE1. Thus, Gic1 could mediate MEN activation by Lte1. However, attempts to show interaction of Gic proteins and Lte1 by yeast two-hybrid, coimmunoprecipitation and in vitro binding failed (not depicted) and Gic1 associated with the bud cortex even in the absence of Lte1 (Fig. 5 D). Moreover, the observation that the
lte1 cdc5-10 phenotype becomes more severe upon deletion of GIC1 and GIC2 excludes such a simple linear pathway. Therefore, we favor the branched pathway outlined in Fig. 7 (steps 1 and 4).
The mitotic spindle and the centrosome also regulate cell cycle progression in other organisms although the molecular mechanisms are much less understood. In fission yeast, a MAPK pathway seems to coordinate spindle positioning and cell cycle progression (Gachet et al., 2001). In mammalian cells, misoriented spindles caused a delay in anaphase onset (O'Connell and Wang, 2000). The centrosome regulates cell cycledependent mitotic exit in animal cells (Piel et al., 2001). It will be interesting to see whether conserved proteins found at the cell cortex, such as Cdc42 and its effectors, are common regulators that coordinate spindle alignment and anaphase onset with cell cycle progression. As a general principal, promoters of mitotic exit may be released from the cell cortex and thereby activated upon spindle elongation in anaphase.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
To analyze cells for a mitotic exit defect, yeast cells with CDC14-GFP were incubated at 30°C for 3 h with 10 µg/ml of -factor to arrest cells in G1 phase of the cell cycle.
-Factor was removed by washing the cells twice with medium (t = 0). Cells were incubated at the indicated temperature. The budding index and nucleolar Cdc14-GFP of fixed cells were determined by phase-contrast and fluorescence microscopy over time. DNA was stained with DAPI.
In vitro binding experiments and two-hybrid analysis
Expression of plasmids encoding GST, His6, and MBP gene fusions in E. coli BL21 DE3 was induced by the addition of 0.5 mM IPTG to L-broth. The cells were incubated for 6 h at 23°C. GST fusion proteins were incubated with glutathione-Sepharose beads (Amersham Biosciences). MBP fusion proteins were presented to amylose resin beads (New England BioLabs, Inc.) and His6 fusions were bound to Ni2+NTA-agarose (QIAGEN). Proteins were affinity purified as recommended by the manufacturers. Protein concentration was determined using protein assay solution (Bio-Rad Laboratories) and was confirmed by PAGE followed by Coomassie blue staining. For in vitro binding experiments, 20 nM of bead-bound protein were incubated with 20 nM of soluble recombinant protein (total volume 1 ml) for 1 h at 4°C in binding buffer (PBS, 1 mM DTT, 5 mM MgCl2, 0.05% NP-40, 100 µM GTP). After three washes with binding buffer, the associated proteins were eluted with sample buffer and analyzed by immunoblotting. Two-hybrid interactions were determined in strain SGY37 with GIC1, BUB2, BFA1, CDC14, and CDC42 subcloned into pMM5 and pMM6 (Geissler et al., 1996).
Immunological techniques and microscopy
Mouse monoclonal anti-GST antibodies and polyclonal rabbit anti-Clb2, anti-Sic1, anti-Tem1, and anti-Tub2 antibodies have been described previously (Pereira et al., 2002). Monoclonal mouse anti-HA (12CA5) and anti-Myc (9E10) antibodies were obtained from Boehringer and mouse monoclonal anti-MBP antibodies were obtained from New England BioLabs, Inc. Secondary antibodies were obtained from Jackson ImmunoResearch Laboratories. Anti-Bub2 and anti-Gic1 antibodies were raised in sheep against the recombinant GST fusion proteins purified from E. coli. Antibodies were affinity purified with recombinant protein coupled to CNBr-Sepharose (Amersham Biosciences). Tem1-9Myc was immunoprecipitated from yeast cell extracts using anti-Myc antibodies coupled to ProA-Sepharose beads.
F-actin was stained with rhodamine-phalloidin (Höfken and Schiebel, 2002). For fluorescence microscopy, Z sequences were collected on an Axiophot microscope (Carl Zeiss MicroImaging, Inc.) controlled by Metamorph software (Universal Imaging Corp.) using a Coolsnap HQ camera (Photometrics). Images were deconvoluted with Huygens software (Scientific Volume Imaging), and colored and merged using Adobe Photoshop.
Online supplemental material
Fig. S1 shows that Gic1 does not disrupt the Net1Cdc14 complex. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200309080/DC1.
![]() |
Acknowledgments |
---|
The work of E. Schiebel is supported by Cancer Research UK.
Submitted: 11 September 2003
Accepted: 9 December 2003
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adames, N.R., and J.A. Cooper. 2000. Microtubule interactions with the cell cortex causing nuclear movements in Saccharomyces cerevisiae. J. Cell Biol. 149:863874.
Adames, N.R., J.R. Oberle, and J.A. Cooper. 2001. The surveillance mechanism of the spindle position checkpoint in yeast. J. Cell Biol. 153:159168.
Bardin, A.J., R. Visintin, and A. Amon. 2000. A mechanism for coupling exit from mitosis to partitioning of the nucleus. Cell. 102:2131.[Medline]
Booher, R.N., R.J. Deshaies, and M.W. Kirschner. 1993. Properties of Saccharomyces cerevisiae wee1 and its differential regulation of p34CDC28 in response to G1 and G2 cyclins. EMBO J. 12:34173426.[Abstract]
Bose, I., J.E. Irazoqui, J.J. Moskow, E.G. Bardes, T.R. Zyla, and D.J. Lew. 2001. Assembly of scaffold-mediated complexes containing Cdc42p, the exchange factor Cdc24p, and the effector Cla4p required for cell cycle-regulated phosphorylation of Cdc24p. J. Biol. Chem. 276:71767186.
Brown, J.L., M. Jaquenoud, M.-P. Gulli, J. Chant, and M. Peter. 1997. Novel Cdc42-binding proteins Gic1 and Gic2 control cell polarity in yeast. Genes Dev. 11:29722982.
Butty, A.C., N. Perrinjaquet, A. Petit, M. Jaquenoud, J.E. Segall, K. Hofmann, C. Zwahlen, and M. Peter. 2002. A positive feedback loop stabilizes the guanine-nucleotide exchange factor Cdc24 at sites of polarization. EMBO J. 21:15651576.
Chen, G.C., Y.J. Kim, and C.S. Chan. 1997. The Cdc42 GTPase-associated proteins Gic1 and Gic2 are required for polarized cell growth in Saccharomyces cerevisiae. Genes Dev. 11:29582971.
Christianson, T.W., R.S. Sikorski, M. Dante, J.H. Shero, and P. Hieter. 1992. Multifunctional yeast high-copy-number shuttle vectors. Gene. 110:119122.[CrossRef][Medline]
Drees, B.L., B. Sundin, E. Brazeau, J.P. Caviston, G.C. Chen, W. Guo, K.G. Kozminski, M.W. Lau, J.J. Moskow, A. Tong, et al. 2001. A protein interaction map for cell polarity development. J. Cell Biol. 154:549571.
Finley, D., K. Tanaka, C. Mann, H. Feldmann, M. Hochstrasser, R. Vierstra, S. Johnston, R. Hampton, J. Haber, J. McCusker, et al. 1998. Unified nomenclature for subunits of the Saccharomyces cerevisiae proteasome regulatory particle. Trends Biochem. Sci. 23:244245.[CrossRef][Medline]
Fraschini, R., E. Formenti, G. Lucchini, and S. Piatti. 1999. Budding yeast Bub2 is localized at the spindle pole bodies and activates the mitotic checkpoint via a different pathway from Mad2. J. Cell Biol. 145:979991.
Gachet, Y., S. Tournier, J.B.A. Millar, and J. Hyams. 2001. A MAP kinase-dependent actin checkpoint ensures proper spindle orientation in fission yeast. Nature. 412:352355.[CrossRef][Medline]
Geissler, S., G. Pereira, A. Spang, M. Knop, S. Souès, J. Kilmartin, and E. Schiebel. 1996. The spindle pole body component Spc98p interacts with the -tubulin-like Tub4p of Saccharomyces cerevisiae at the sites of microtubule attachment. EMBO J. 15:38993911.[Abstract]
Geymonat, M., A. Spanos, S.J. Smith, E. Wheatley, K. Rittinger, L.H. Johnston, and S.G. Sedgwick. 2002. Control of mitotic exit in budding yeast. In vitro regulation of Tem1 GTPase by Bub2 and Bfa1. J. Biol. Chem. 277:2843928445.
Geymonat, M., A. Spanos, P.A. Walker, L.H. Johnston, and S.G. Sedgwick. 2003. In vitro regulation of budding yeast Bfa1/Bub2 GAP activity by Cdc5. J. Biol. Chem. 278:1459114594.
Glotzer, M., A.W. Murray, and M.W. Kirschner. 1991. Cyclin is degraded by the ubiquitin pathway. Nature. 349:132138.[CrossRef][Medline]
Höfken, T., and E. Schiebel. 2002. A role for cell polarity proteins in mitotic exit. EMBO J. 21:48514862.
Hu, F., Y. Wang, D. Liu, Y. Li, J. Qin, and S.J. Elledge. 2001. Regulation of the Bub2/Bfa1 GAP complex by Cdc5 and cell cycle checkpoints. Cell. 107:655665.[Medline]
Jaspersen, S.L., J.F. Charles, R.L. Tinker-Kulberg, and D.O. Morgan. 1998. A late mitotic regulatory network controlling cyclin destruction in Saccharomyces cerevisiae. Mol. Biol. Cell. 9:28032817.
Jensen, S., M. Geymonat, A.L. Johnson, M. Segal, and L.H. Johnston. 2002. Spatial regulation of the guanine nucleotide exchange factor Lte1 in Saccharomyces cerevisiae. J. Cell Sci. 115:49774991.[CrossRef][Medline]
Knop, M., K. Siegers, G. Pereira, W. Zachariae, B. Winsor, K. Nasmyth, and E. Schiebel. 1999. Epitope tagging of yeast genes using a PCR-based strategy: more tags and improved practical routines. Yeast. 15:963972.[CrossRef][Medline]
Longtine, M.S., A. McKenzie, D.J. Demarini, N.G. Shah, A. Wach, A. Brachat, P. Philippsen, and J.P. Pringle. 1998. Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast. 14:953961.[CrossRef][Medline]
Maekawa, H., T. Usui, M. Knop, and E. Schiebel. 2003. Yeast Cdk1 translocates to the plus end of cytoplasmic microtubules to regulate bud cortex interactions. EMBO J. 22:438449.
Mah, A.S., J.J. Jang, and R.J. Deshaies. 2001. Protein kinase Cdc15 activates the Dbf2-Mob1 kinase complex. Proc. Natl. Acad. Sci. USA. 98:73257330.
Mitchell, D.A., L. Farh, T.K. Marshall, and R.J. Deschenes. 1994. A polybasic domain allows nonprenylated Ras proteins to function in Saccharomyces cerevisiae. J. Biol. Chem. 269:2154021546.
O'Connell, C.B., and Y. Wang. 2000. Mammalian spindle orientation and position respond to changes in cell shape in a dynein-dependent fashion. Mol. Biol. Cell. 11:17651774.
Pereira, G., and E. Schiebel. 2001. The role of the yeast spindle pole body and the mammalian centrosome in regulating late mitotic events. Curr. Opin. Cell Biol. 13:762769.[CrossRef][Medline]
Pereira, G., T. Höfken, J. Grindlay, C. Manson, and E. Schiebel. 2000. The Bub2p spindle checkpoint links nuclear migration with mitotic exit. Mol. Cell. 6:110.[Medline]
Pereira, G., T.U. Tanaka, K. Nasmyth, and E. Schiebel. 2001. Modes of spindle pole body inheritance and segregation of the Bfa1p/Bub2p checkpoint protein complex. EMBO J. 20:63596370.
Pereira, G., C. Manson, J. Grindlay, and E. Schiebel. 2002. Regulation of the Bfa1pBub2p complex at spindle pole bodies by the cell cycle phosphatase Cdc14p. J. Cell Biol. 157:367379.
Piel, M., J. Nordberg, U. Euteneuer, and M. Bornens. 2001. Centrosome-dependent exit of cytokinesis in animal cells. Science. 291:15501553.
Pryciak, P.M., and F.A. Huntress. 1998. Membrane recruitment of the kinase cascade scaffold protein Ste5 by the G ß complex underlies activation of the yeast pheromone response pathway. Genes Dev. 12:26842697.
Schwab, M., A.S. Lutum, and W. Seufert. 1997. Yeast Hct1 is a regulator of Clb2 cyclin proteolysis. Cell. 90:683693.[Medline]
Schwob, E., T. Bohm, M.D. Mendenhall, and K. Nasmyth. 1994. The B-type cyclin kinase inhibitor p40 (Sic1) controls the G1 to S transition in Saccharomyces cerevisiae. Cell. 79:233244.[Medline]
Seshan, A., A.J. Bardin, and A. Amon. 2002. Control of Lte1 localization by cell polarity determinants and Cdc14. Curr. Biol. 12:20982110.[CrossRef][Medline]
Sherman, F. 1991. Getting started with yeast. Methods Enzymol. 194:321.[Medline]
Shirayama, M., Y. Matsui, K. Tanaka, and A. Toh-e. 1994a. Isolation of a CDC25 family gene, MSI2/LTE1, as a multicopy suppressor of ira1. Yeast. 10:451461.[Medline]
Shirayama, M., Y. Matsui, and A. Toh-e. 1994b. The yeast TEM1 gene, which encodes a GTP-binding protein, is involved in termination of M-phase. Mol. Cell. Biol. 14:74767482.[Abstract]
Shou, W., J.H. Seol, A. Shevchenko, C. Baskerville, D. Moazed, W.S. Chen, J. Jang, A. Shevchenko, H. Charbonneau, and R. Deshaies. 1999. Exit from mitosis is triggered by Tem1-dependent release of the protein phosphatase Cdc14 from nucleolar RENT complex. Cell. 97:233244.[Medline]
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. 122:1927.
Stegmeier, F., R. Visintin, and A. Amon. 2002. Separase, polo kinase, the kinetochore protein Slk19, and Spo12 function in a network that controls Cdc14 localization during early anaphase. Cell. 108:207220.[Medline]
Uetz, P., L. Giot, G. Cagney, T.A. Mansfield, R.S. Judson, J.R. Knight, D. Lockshon, V. Narayan, M. Srinivasan, P. Pochart, et al. 2000. A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae. Nature. 403:623627.[CrossRef][Medline]
Visintin, R., S. Prinz, and A. Amon. 1997. CDC20 and CDH1: a family of substrate-specific activators of APC-dependent proteolysis. Science. 278:460463.
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. 2:709718.[Medline]
Visintin, R., E.S. Hwang, and A. Amon. 1999. Cfi1 prevents premature exit from mitosis by anchoring Cdc14 phosphatase in the nucleolus. Nature. 398:818823.[CrossRef][Medline]
Visintin, R., F. Stegmeier, and A. Amon. 2003. The role of polo kinase Cdc5 in the FEAR and mitotic exit networks. Mol. Biol. Cell. 14:44864498.
Wang, Y., T. Shirogane, D. Liu, W. Harper, and S.J. Elledge. 2003. Exit from exit: resetting the cell cycle through Amn1 inhibition of G protein signalling. Cell. 112:697709.[Medline]
Yoshida, S., R. Ichihashi, and A. Toh-e. 2003. Ras recruits mitotic exit regulator Lte1 to the bud cortex in budding yeast. J. Cell Biol. 161:889897.