Function of Lte1-interacting Proteins in the Spindle Position Checkpoint
Null mutants lacking the map's proximate Lte1-interacting proteins were tested for loss of integrity of the spindle position checkpoint. Time-lapse fluorescence movies of the time course of mitosis in single living cells expressing GFP-tubulin were performed, as described (Castillon et al., 2003
; A). Live movie assays allow one to follow individual cells, rather than observe the average behavior of a synchronized population, providing definitive information as to whether and when mitotic exit occurs in cells with abnormal or delayed positioning of the mitotic spindle. To increase the fraction of cells that delay movement of the spindle into the mother-bud neck, we inactivated dynein by deleting the gene for the dynactin component Arp1. To analyze the data, an observer viewed the movie and identified all cells with late-anaphase (long) spindles in the mother. The observer followed each cell forward in time and recorded whether the cell exited mitosis and the time of mitotic exit, if it occurred. Mitotic exit was marked by breakdown of the fluorescent spindle. The spindle buckles, breaks in the middle, and then spindle microtubules disassemble. Many cells were followed through the end of and into the next cell cycle, and in every case, breakdown of the spindle was followed by a full course of events, including completion of cell division, new bud formation and SPB duplication. A number of other cellular events compose the processes of mitotic exit and the ensuing cell division (Juang et al., 1997
; Adames et al., 2001
Figure 2. Spindle position checkpoint in cells lacking Lte1-interacting proteins. (A) Lte1-interacting proteins are required for checkpoint integrity. arp1Δ GFP-TUB1 cells with the indicated additional mutation were assayed for checkpoint integrity. Cells (more ...)
Otherwise wild-type arp1
Δ cells with late-anaphase spindles in the mother remained arrested indefinitely, whereas mitotic exit occurred promptly in mutants lacking spindle position checkpoint components (A), as seen in previous studies (Adames et al., 2001
; Castillon et al., 2003
). Checkpoint integrity was calculated as the number of cells remaining arrested divided by the sum of the number of arrested cells plus the number of cells undergoing mitotic exit with the spindle in the mother. In some cells, the late-anaphase spindle was able to enter the neck. When this happened, mitotic exit ensued promptly, creating two cells with single nuclei. The frequency of this correction of spindle position was 6–8%, with similar values in wt, bub2
Δ, and msl1
Δ strains. These cells were not included in the checkpoint integrity calculation.
As a positive control in this assay, GAP-deficient bub2Δ arp1Δ cells displayed 0% checkpoint integrity, meaning that every late-anaphase spindle in the mother underwent mitotic exit in the mother. In addition, mitotic exit occurred promptly, which was defined as a time less than the mean plus 2 SDs of the time for normal mitotic exit.
One Lte1 interactor, Msl1, identified by a high throughput two-hybrid screen, was not previously implicated in the cell cycle (Fromont-Racine et al., 1997
). Deletion of MSL1 in an arp1
Δ background caused a substantial decrease of checkpoint integrity, to a value of 59%. The tpd3
Δ mutant was normal. We also found loss of SPC integrity in null mutants lacking Ras1, Ras2, Cla4, or Kel1, each of which have been implicated in Lte1 function in some way in previous studies (Hofken and Schiebel, 2002
; Seshan et al., 2002
; Yoshida et al., 2003
; Seshan and Amon, 2005
Msl1 has been characterized as the U2B″ subunit of the U2 small nuclear ribonucleoprotein (snRNP; Tang and Cai, 1996
). To assess the potential role of RNA splicing or snRNPs in the checkpoint, we assayed lea1
null mutants, which lack the U2A′ subunit of the U2 snRNP. Lea1 interacts biochemically with Msl1 (also known as Yib9), and msl1
null mutants have similar phenotypes with respect to U2 assembly, splicing activity, and growth (Caspary and Seraphin, 1998
). A lea1
Δ mutant had full integrity of the spindle position checkpoint (Supplementary Figure 1), as did a mud1
Δ mutant, which lacks the U1-A component of the U1 snRNP (Liao et al., 1993
; Neubauer et al., 1997
Our hypothesis was that the checkpoint works through upstream regulators to inhibit Lte1 when the spindle has not yet entered the neck. In null mutants lacking such regulators, Lte1 would then be more active than normal, which would cause mitotic exit to fail to wait. If the checkpoint functions of these Lte1-interacting proteins are upstream of Lte1, then the loss of checkpoint in the null mutants should be suppressed by loss of Lte1. We combined the null mutations with an lte1Δ mutation. For the msl1Δ mutant, the checkpoint phenotype was completely suppressed by the lte1Δ mutation. The checkpoint defects of mutants lacking Ras1, Ras2, and Cla4, also depended on Lte1 (B). Bub2/Bfa1, in its role as a putative GAP for Tem1, might not be expected to depend on Lte1 in this assay. Indeed, the checkpoint phenotype of a bub2Δ mutant was not suppressed by the addition of an lte1Δ mutation (B). Thus, Msl1 appears to function upstream of Lte1 with respect to checkpoint function.
To further characterize the role of Msl1 and other Lte1-interactors, we tested checkpoint integrity at low temperature. During the normal course of mitosis at 30°C, Tem1 can be activated and mitotic exit can occur even in the absence of Lte1, probably because the intrinsic GTP exchange activity of Tem1 is sufficiently high at this temperature (Geymonat et al., 2002
). In the absence of Lte1, inhibition of Bub2/Bfa1 GAP activity is presumed to be the regulatory event needed to activate Tem1 and drive mitotic exit. However, at low temperature, where the intrinsic GTP exchange rate of Tem1 is lower, Lte1 is required for mitotic exit, and lte1
Δ mutants fail to grow because they arrest in anaphase (Shirayama et al., 1994b
If Msl1 inhibits Lte1, then an msl1
Δ mutant should have increased Lte1 activity, and thus, in the cold, msl1
Δ cells might exit from mitosis instead of arrest in mitosis. To test this hypothesis, we counted multinucleated cell bodies, as an indicator of mitotic exit and checkpoint failure, in arp1
Δ cells at 12 or 30°C and (C). For msl1
Δ, the value was 2.5-fold higher at 12 than at 30°C, and similar results were seen for ras1
Δ and ras2
Δ mutants. A bub2
Δ mutant was also included, as a control (D), and its value for multinucleate cell bodies was similar to that of msl1
Δ. At 30°C, the bub2
Δ mutation is known to cause a complete loss of the checkpoint, by movie analysis (Castillon et al., 2003
; B). Movie analysis is not practical at 12°C, but the similarity of phenotypes for msl1
Δ and bub2
Δ in this assay, in the cold, suggests that Msl1, working through Lte1, may have a critical role in delaying mitotic exit.
In these assays, loss of Cla4, Ras1, or Ras2 also promoted mitotic exit in an arp1
Δ background, and this phenotype was suppressed by loss of Lte1 in each case (, A and B). The simplest interpretation of these results is that Cla4, Ras1, and Ras2 inhibit Lte1. However, this seems paradoxical because, in previous studies, cla4
Δ and lte1
Δ mutants had a similar phenotype of poor growth in the cold (Hofken and Schiebel, 2002
; Seshan et al., 2002
), and cla4, ras1, ras2
, and lte1
show synthetic lethality with FEAR mutations (Stegmeier et al., 2002
; Goehring et al., 2003
; Yoshida et al., 2003
). These results suggest that Lte1 and the three other proteins function in the same direction, not opposing directions (Hofken and Schiebel, 2002
; Seshan et al., 2002
). We confirmed that a cla4
Δ single mutant, which is otherwise wild type, grows poorly in the cold in our strain background (data not shown), and we confirmed the cla4
Δ checkpoint phenotype by rescue with CLA4
on a plasmid (A). Several considerations may account for the apparent discrepancy—the movie assay for mitotic exit is quite different and far more specific than the assay for growth on plates in the cold, the assay for mitotic exit is done in a background without dynein function, and Cla4 is a kinase known to have functions in other pathways unrelated to mitosis (Gulli et al., 2000
; Gladfelter et al., 2004
In previous studies, mutants with unstable cytoplasmic microtubules suffered a loss of the spindle position checkpoint, and movies suggested that loss of cytoplasmic microtubule contact with the mother/bud neck was the common event that preceded mitotic exit (Adames et al., 2001
). These observations suggested that microtubule/neck interaction might activate the checkpoint and delay mitotic exit (Adames et al., 2001
). To determine whether the premature or “inappropriate” mitotic exit in msl1
Δ cells was due to a primary loss of checkpoint activity or was instead secondary to a defect in microtubule dynamics, we followed the dynamics of cytoplasmic microtubules during mitotic exit. This experiment required image collection at short time intervals over a long time duration, which is challenging (A, Supplementary Movie 1).
Figure 3. Loss of spindle position checkpoint integrity in an msl1 mutant is not coupled with defects in microtubule dynamics. (A) Frames at 2-min intervals from a time-lapse movie of a representative msl1Δ arp1Δ GFP-TUB1 cell (Supplementary Movie (more ...)
In 6 of 13 cases of spindle breakdown in the mother cell in an msl1 mutant, we saw that cytoplasmic microtubules were present in the neck when the spindle broke down (B). In several cases, the microtubule appeared to be cleaved in two at the neck by the process of cytokinesis. In the other 7 of 13 cases, the cytoplasmic microtubules withdrew from the neck at the same time as the spindle broke down. These results are consistent with msl1Δ cells having a primary defect in the checkpoint, as opposed to a defect secondary to microtubules.
Only about half of the mutant cells with a mispositioned spindle proceeded to mitotic exit with the spindle in the mother; in the other half, the cell cycle was arrested. To understand the difference between these populations, we asked how rapidly mitotic exit occurred. In the movie analysis above, cells with mispositioned anaphase spindles were chosen for observation, raising the possibility that the cells had been arrested in mitosis for a significant period of time before the movie began. To test this possibility, we repeated the experiment, choosing cells in early anaphase allowing one to follow the entire time course of mitosis (Supplementary Figure 2). As usual, a dynein-deficient background (arp1Δ) was used, so that movement of the spindle into the neck was delayed in about one-third of cells. In an otherwise wt cell in which the spindle entered the neck normally, mitosis took an average of 25 min (timed from the start of spindle elongation to spindle breakdown). For msl1Δ, ras1Δ, ras2Δ, and cla4Δ cells in which the spindle entered the neck normally, the time was similar. However, when spindles did not enter the neck and mitosis occurred in the mother cell, mitosis in wild-type cells was significantly longer. In contrast, msl1Δ, ras1Δ, and ras2Δ mutants did not significantly delay mitosis when the spindle stayed in the mother. The cla4Δ mutant had a small but significant delay compared with wild type. In other words, in mutant cells in which the checkpoint failed, the timing of mitosis was normal. Thus, these cells did not display any delay before the checkpoint failed, indicating a complete loss in the checkpoint in those individual cells.
To further investigate the timing of mitosis, we returned to the datasets of movies in which cells with mispositioned spindles in the mother were selected for analysis. We measured the time required for breakdown of the spindle in the mother. These data are shown as histograms in Supplementary Figure 3. Otherwise wild-type arp1Δcells, with mispositioned spindles in the mother, usually remained arrested throughout the movie, usually ~90 min. When spindles in the mother of otherwise wild-type cells did break down, they did so after being arrested for a highly variable length of time, and this generally followed loss of cytoplasmic microtubules from the neck or abortive entry of the spindle into the neck. When spindles in the mother of msl1Δ, ras1Δ, ras2Δ, and cla4Δ cells broke down, the majority of cells did so in 20–30 min, similar to the time for mitosis of a spindle entering the neck of an otherwise wild-type cell. Only a minority of cells in each mutant significantly delayed mitosis before spindle breakdown. These data are also consistent with mitotic exit occurring with normal rapidity when the checkpoint is lost in mutant cells. The results do not contradict the observation that the checkpoint mechanism fails in only ~40% of cells. The mechanism is apparently intact in the other ~60% of cells, which do arrest. Therefore, the mechanism may involve a level of cooperativity or positive feedback.
Linking Lte1 Regulation with Microtubule-Cortex Interactions
To further investigate the role of Msl1 activating the checkpoint, we returned to the proteomic map and looked for connections of Msl1 with proteins of the bud neck and the cytoskeleton (B). The map shows that Msl1 binds Atc1 according to a high-throughput two-hybrid assay, which is named A
omplex 1 for its ability to interact, by two-hybrid assay, with the protein Aip3, also known as Bud6 (Freedman et al., 2000
; Amberg and Haarer, SUNY Upstate, personal communication, 2007). We will refer to Aip3/Bud6 as Bud6 for simplicity. Bud6 is located in a ring at the bud neck and as puncta on the bud cortex (Huisman et al., 2004
; Huisman and Segal, 2005
). In both locations, Bud6 has been observed to interact with cytoplasmic microtubules, based on two-color movies (Huisman and Segal, 2005
A previous study of Bud6 found multinucleated cells in asynchronous cultures of bud6
null mutants and observed mitotic exit in some cells with late-anaphase spindles in the mother (Huisman et al., 2004
). We uncovered bud6
Δ mutants in a screen for spindle position checkpoint mutants (Heil-Chapdelaine et al.
, 2002; unpublished data), as described in Materials and Methods.
We analyzed atc1 and bud6 null mutants as described above for msl1Δ. Based on movie analysis in an arp1 background, atc1Δ and bud6Δ mutants had decreased values for checkpoint integrity, with values similar to that of the msl1Δ mutant (A). Deletion of LTE1 strongly suppressed the checkpoint phenotypes of the atc1Δ and bud6Δ mutants, as seen for msl1 (A). Placing cells in the cold, we found an increase in the number of multinucleate cell bodies in bud6 and atc1Δ mutants, at a level similar to that seen for msl1Δ and bub2Δ (, C and D).
Figure 4. Genetic analysis of MSL1, ATC1, and BUD6 in the spindle position checkpoint. (A) bud6 and atc1 mutants display decreased checkpoint integrity, with values similar to that of msl1 (p = 0.004, 0.009, and 0.002, respectively, with respect to wild type). (more ...)
To extend the genetic analysis, we analyzed double null mutants (B). Deleting BUD6 did not enhance or suppress the checkpoint phenotype of atc1Δ or msl1Δ mutants, suggesting that ATC1, MSL1, and BUD6 lie in the same genetic pathway with respect to checkpoint function. Additionally, like msl1, ras1, and ras2 mutants, mitotic exit in the mother occurred at the same rate as mitotic exit in the mother-bud neck (Supplementary Figures 2 and 3).
As a further test of the existence of a pathway, we asked whether overexpression of one gene was able to suppress the checkpoint phenotype in a null mutant lacking another gene (). Based on the physical interactions in the database, one might hypothesize that Bud6 lies upstream of Atc1, Atc1 is upstream of Msl1, and Msl1 inhibits Lte1. To test this simple model, we overexpressed each gene in a strain lacking the other genes, in pair wise combinations. The most straightforward result would be that overexpression of a gene that is downstream of a second gene suppresses the phenotype of a mutant lacking the second gene. The assay was counting multinucleate cell bodies in asynchronous cultures of strains with an arp1Δ background. The checkpoint defect of bud6Δ cells was completely suppressed by overexpression of ATC1 or MSL1 (). Deletion of LTE1 also suppressed the checkpoint phenotype of bud6Δ, atc1Δ, and msl1Δ mutants, confirming the results above with movie analysis. The checkpoint defect of atc1Δ cells was completely suppressed by overexpression of MSL1 and partially suppressed by overexpression of BUD6. The checkpoint defect of msl1Δ cells was not suppressed by overexpression of BUD6 or ATC1. No overexpression allele caused a loss of checkpoint in lte1Δ cells (). Furthermore, no gene deletion was able to suppress the checkpoint defect of cells overexpressing LTE1. All the results, with the sole exception of partial, not full, suppression of atc1Δ by BUD6, are consistent with Bud6, Atc1, and Msl1 acting in an ordered pathway to inhibit Lte1 function. This order revealed by this assay is consistent with the order of the protein interactions in the two-hybrid analysis (B). The results do not exclude the possible existence of simultaneous multisubunit interactions.
Predicted results of overexpression suppression analysis of Lte1 regulators in the checkpoint pathway
Observed results of overexpression suppression analysis of Lte1 regulators in the checkpoint pathway
Atc1-GFP and Msl1-GFP were both found throughout the cytoplasm in the mother and bud, with a uniform distribution (Supplementary Figure 4). Msl1-GFP was slightly concentrated in the nucleus, and it appeared to be excluded from the vacuole. The fluorescence signal of Atc1-GFP and Msl1-GFP was significantly higher than the background fluorescence of a cell not expressing GFP. The uniform cytoplasmic distribution of these proteins was similar to that of plain GFP, which was excluded from the nucleus and not concentrated anywhere. The localization of Atc1 and Msl1 was similar in cells at different stages of the cell cycle.
The Cellular Role of Bud6 in Spindle Position Checkpoint Integrity
Bud6 forms a ring at the neck and foci in the bud cortex in late mitosis, where it captures microtubule ends (Huisman and Segal, 2005
). We asked whether loss of microtubule capture might be responsible for the checkpoint defect in bud6
Δ mutants. First, we colocalized microtubules and Bud6 in anaphase cells in which the checkpoint was activated due to the presence of a late-anaphase spindle in the mother. Microtubule ends, seen by CFP-Tub1, were often colocalized with cortical foci of GFP-Bud6 (A), as predicted.
Figure 5. Microtubule capture and the spindle position checkpoint. (A) Microtubule plus ends colocalize with Bud6 foci at the bud cortex (left) and at the mother-bud neck (right) when cells have anaphase spindles in the mother and the checkpoint is active. Representative (more ...)
To test the significance of the Bud6-microtubule interaction and to identify other molecular components involved in the interaction, we examined the colocalization of microtubule ends with Bud6 at cortical foci or neck rings in mutants lacking cytoplasmic microtubule-binding proteins (B). In 73% of wild-type cells, microtubule ends were observed to be colocalized with Bud6 at the neck or at cortical foci. This value was reduced to 5% in a kinesin/kip2Δ mutant, and 31% in a CLIP170/bik1Δ mutant. All other mutants tested had normal values, including tubulin-folding cofactor B/alf1Δ, EB1/bim1Δ, coronin/crn1Δ, kinesin/kip3Δ, p150/Glued/nip100Δ, ase1Δ, atg8Δ, and mhp1Δ. Thus, the kinesin Kip2 and, to a lesser extent, CLIP170/Bik1, are specifically required for Bud6-mediated microtubule capture.
To test whether microtubule/Bud6 interactions are required for the checkpoint, we assayed checkpoint integrity in kip2Δ and bik1Δ strains because these mutants have poor microtubule/Bud6 interactions. The kip2Δ mutant displayed decreased checkpoint integrity, at a level similar to that of a bud6Δ mutant (C). A kip2Δ bud6Δ double mutant had the same value that the single mutants did, suggesting that the two proteins participate in the same process. bik1Δ strains displayed an intermediate value for checkpoint integrity, consistent with the intermediate loss of Bud6/microtubule colocalization in this mutant. Therefore, microtubule capture by the neck or cortex, involving the participation of Bik1, Kip2, and Bud6, correlates with and appears to be necessary for activation of the checkpoint.
Mutants lacking Bik1 and Kip2 have short cytoplasmic microtubules(Berlin et al., 1990
; Carvalho et al., 2004
), suggesting that this trait might account for the observed decrease in cortical Bud6/microtubule interactions. To test this possibility, we quantified the frequency of colocalization of microtubules with the bud cortex at sites away from Bud6 foci in wild-type, bik1
Δ, and kip2
Δ cells (Supplementary Figure 5). No significant differences were found, indicating that microtubules in the mutants are able to touch the cortex and thus have the opportunity to make productive capture interactions. We also addressed this issue by deleting the kinesin kip3
in the kip2
mutant, which has been shown to restore microtubule length (Cottingham and Hoyt, 1997
). In our strain background, the kip3
Δ mutant had the same low value of microtubule/Bud6 interactions as seen in the single kip2
mutant (B), despite having qualitatively longer cytoplasmic microtubules. Therefore, kip2
Δ and bik1
Δ mutations appear to have a direct effect on microtubule interactions with the cortex at Bud6 foci.
Because Kip2 appears to connect microtubules to Bud6 in the cell, we hypothesized that the KIP2 gene might lie upstream of the BUD6 gene in the genetic pathway for regulation of LTE1. To test this hypothesis, we performed suppression analysis, as above. The kip2Δ checkpoint phenotype was suppressed by overexpression of BUD6, ATC1, or MSL1 (). Conversely, overexpression of KIP2 in bud6Δ, atc1Δ, msl1Δ, and lte1Δ mutants did not suppress the checkpoint phenotype of any of these mutants (). These results place KIP2 upstream of BUD6 in the checkpoint pathway, as defined by genetic interactions.
To test the specificity of the role of Bud6 in the spindle position checkpoint, as opposed to other checkpoints, we assayed bud6Δ mutants for defects in the bud morphogenesis and DNA damage checkpoints by assaying growth in latrunculin A and hydroxyurea, respectively (Supplementary Figure 6). Both checkpoints were intact.
In addition to roles in microtubule capture, BUD6
is also known to be important for bud-site selection and actin cable formation (Amberg et al., 1997
; Moseley et al., 2004
). We found that bud1
mutants, which are completely defective in bud-site selection, showed no loss of spindle position checkpoint integrity by movie analysis (Supplementary Figure 7A). In addition, loss of actin cables, caused by mutations in genes encoding formins, fimbrin, or tropomyosin, did not cause a significant loss of checkpoint integrity (Supplementary Figure 7B). These results suggest that the role of Bud6 in the spindle position checkpoint is based on its role in microtubule capture.
Lte1 Localization in Checkpoint Mutants
In the events leading up to mitotic exit, Tem1 accumulates at the daughter-bound SPB, and Lte1 localizes to the bud cortex (Bardin et al., 2000
; Molk et al., 2004
). These observations led to a model in which movement of the SPB into the bud allows interaction of Tem1 with Lte1 (Bardin et al., 2000
). In support of this model, spindle position checkpoint failure has been observed to result from septin mutations that allow Lte1 to cross the neck from the bud into the mother (Castillon et al., 2003
). In the mother, this ectopic Lte1 is presumed to activate Tem1 and thus the MEN (Shirayama et al., 1994b
Bud6 is a component of the mother-bud neck, so we considered whether the checkpoint defect of a bud6
Δ mutant might be due a defective neck leading to the presence of ectopic Lte1 in the mother. In a previous study, Lte1 was observed to be localized normally in a bud6
mutant (Jensen et al., 2002
). We confirmed this result with Lte1-3GFP and found that Lte1 localization appeared normal in atc1
Δ, and kip2
Δ cells as well (data not shown). To address the issue of Lte1 in the mother directly, we digitally imaged and quantified the fluorescence of Lte1-3GFP in the mother cytoplasm. In a wild-type cell, the level of this fluorescence is not greater than the fluorescence associated with a control cell that does not express any GFP (Castillon et al., 2003
). We found that bud6
Δ cells have no more Lte1-3GFP fluorescence in the mother than do wild-type cells. As a positive control, a sep7
Δ mutant had significantly increased levels, as seen previously (Castillon et al., 2003
As a more stringent test for the diffusion of a small amount of Lte1 from the bud into the mother of bud6Δ cells, at a level undetectable by steady-state fluorescence imaging, we used fluorescence loss in photobleaching (FLIP; , C and D). We photobleached a portion of a mother cell and quantified the fluorescence in the bud. Sequential rounds of photobleaching and imaging were performed. If Lte1-3GFP was present in the mother, it should have been bleached, leading to a loss of fluorescence intensity in the bud over time. Wild-type and bud6Δ cells gave similar results, with only a small loss of fluorescence after 10 rounds of photobleaching, an amount consistent with the degree of photobleaching over the entire field. As a positive control, in sep7Δ cells, the bud fluorescence fell to background levels after three rounds of photobleaching.
Figure 6. Lte1 localization and dynamics. (A) Lte1-3GFP is excluded from the mother in bud6 mutants. Representative images of Lte1-3GFP small- and large-budded cells are shown. The mother cell cytoplasm of sep7Δ cells is brighter than that of wt or bud6 (more ...)
These results suggest that the checkpoint phenotype of bud6
Δ cells is not due to mislocalization of Lte1 in the mother. Furthermore, they suggest that, in the bud6
mutant, Tem1 activation can occur with Lte1 confined to the bud and with the Tem1-rich daughter-bound SPB still in the mother. A possible resolution of this apparent paradox is provided by the observation that Tem1 at the SPB rapidly exchanges with a cytoplasmic pool of Tem1 (Molk et al., 2004
). Based on this and other findings, Molk and colleagues proposed a model in which the cytoplasmic pool of Tem1 is activated by Lte1 in the bud (Molk et al., 2004
The relationship of the cortical localization of Lte1 to the activity state of Lte1 in the cell is not well understood. Lte1 is lost from the bud cortex at the end of the cell cycle, assuming a diffuse distribution in the cytoplasm, which suggests that cortical localization may influence Lte1 activity (Jensen et al., 2002
; Seshan et al., 2002
). The cortical localization of Lte1 requires Ras2 (Yoshida et al., 2003
), Cla4 (Hofken and Schiebel, 2002
), and Kel1 (Seshan et al., 2002
). Here, ras1, ras2
, and cla4
mutants had decreased checkpoint integrity, in the dynein-deficient background (). Their phenotypes were suppressed by the loss of Lte1, and they were not enhanced by the loss of Bud6 (data not shown), suggesting that these proteins influence mitotic exit via Lte1.