PDS5 is required for maintenance of cohesion in mitosis, but not for establishment
Previously, we demonstrated that pds5
mutants exhibit precocious sister dissociation at CEN
-proximal and -distal loci when arrested at nonpermissive temperature in mitosis by treatment with Nz (Hartman et al., 2000
). Furthermore, we showed that pds5
mutants retain cohesion if arrested with Nz at permissive temperature, but rapidly lose cohesion when transferred to nonpermissive temperature, indicating that Pds5p is required to maintain cohesion during metaphase. These experiments could not determine whether budding yeast Pds5p is required for establishment. In Schizosaccharomyces pombe
mutants have normal cohesion after S phase, but exhibit precocious sister dissociation when arrested in G2 phase or mitosis (Tanaka et al., 2001
). Thus, fission yeast Pds5p is required for cohesion maintenance, but not for establishment. To test whether this is also the case in budding yeast, we assessed cohesion in synchronously growing pds5
Wild-type and pds5
mutants were grown in YEPD at 23°C, arrested in S phase using hydroxyurea (HU), shifted to 37°C, and then released into fresh 37°C YEPD containing α-factor (see Materials and methods). By this regimen, cells progress synchronously through S phase and mitosis at the nonpermissive temperature for pds5
mutants, and then arrest in G1 phase. To monitor cohesion, strains contained a Tet operator array integrated at the URA3
locus and also expressed a Tet repressor–GFP fusion protein. Cell cycle progression was monitored by FACS®
analysis and Western blot of Pds1p levels. Pds1p is a negative regulator of the metaphase–anaphase transition, and its destruction can be used as a molecular marker for anaphase onset (Cohen-Fix et al., 1996
; Yamamoto et al., 1996a
). HU-arrested cells at 37°C (t = 0) have unreplicated DNA, so both wild-type and pds5-2
cells had only one GFP signal (). Both wild-type and pds5
cells completed DNA replication by 30 min after release from HU ( B). As expected, few wild-type cells had two GFP signals (separated sisters) because of sister chromatid cohesion. Similarly, few pds5-2
cells had two GFP signals, indicating that sister chromatid cohesion had been established and was still present on most chromosomes. In contrast, cohesin complex mutants exhibited significant loss of cohesion after S phase (Michaelis et al., 1997
). By 45 min, the number of wild-type and pds5-2
cells with two GFP signals increased significantly ( A). In wild-type cells, the sisters separated as a consequence of normal anaphase, as evidenced by decreased Pds1p levels ( C) and the location of CEN
-proximal GFP signals at the leading edges of the separating DNA masses ( D, left). In contrast, Pds1p levels remained high in pds5-2
cells from 45–90 min, even though most cells had two GFP signals (). The separated GFP signals in these cells remained close together ( D, middle). These results indicate that precocious sister dissociation had occurred in pds5
cells and had activated the mitotic checkpoint. Finally, by 120 min in pds5-2
cells, Pds1p levels had decreased and anaphase cells appeared (; right). Similar results were observed with pds5-1
cells (unpublished data). Thus, in pds5
mutants, cohesion is established, but sisters undergo precocious dissociation before anaphase. Therefore, as in fission yeast, budding yeast Pds5p is not required for cohesion establishment, but is required for its maintenance during mitosis.
Figure 1. Characterization of cell cycle and cohesion defects of pds5 cells. Wild-type (VG2450-7A and VG2390-37A) and pds5-2 (VG2456-5C and VG2416-12A) haploids released from S phase (HU arrest) at 37°C. (A) Percentage of cells with two GFP signals. The (more ...)
SMT4 suppresses the temperature sensitivity of pds5 mutants
To gain insight into the role of Pds5p in cohesion, we screened for high copy suppressors of the temperature sensitivity of pds5-1 cells (see Materials and methods). High copy plasmids bearing the SMT4 gene (2μ SMT4) suppressed the temperature sensitivity of pds5-1 cells to an extent comparable to PDS5 itself ( A). High copy SMT4 also suppressed haploid pds5-2 and pds5-3 mutant strains, although suppression was weaker in pds5-3 cells. SMT4 present on a low copy CEN vector also strongly suppressed all three pds5 alleles ( B). These results demonstrate that the temperature sensitivity of pds5 mutants is exquisitely sensitive to SMT4 levels.
Figure 2. SMT4 suppresses pds5 mutant temperature sensitivity. (A) Effect of high copy SMT4 (2μ URA3 vector) on the temperature-sensitive phenotype of pds5 mutants. Haploid strains pds5-1 (VG986-5B), pds5-2 (VG987-5C), and pds5-3 (VG988-1C) containing 2μ (more ...)
The fact that SMT4
robustly suppressed pds5
cells suggested that SMT4
either directly affects a property of the mutant protein or bypasses the need for Pds5p function. To distinguish between these possibilities, we asked whether SMT4
could suppress a deletion of PDS5.
is an essential gene in budding yeast (Hartman et al., 2000
), we used a plasmid shuffle assay to test for suppression of a pds5
-null allele (pds5
; see Materials and methods). A haploid pds5
strain was kept viable by a plasmid-borne copy of PDS5
(pTH10; PDS5 CEN LEU2
). We assayed the ability of high copy plasmid pTH40 (2μ SMT4 TRP1
) to enable plasmid pTH10 loss. After 40 generations of growth in nonselective media, the pTH10 reporter was never lost from cells bearing pTH40 (2μ SMT4 TRP1
). For positive and negative controls, we assayed high copy plasmids pTH39 (2μ PDS5 TRP1
) and YEplac112 (2μ TRP1
), respectively. As expected, the pTH10 reporter plasmid was readily lost from cells bearing pTH39 because it provides Pds5p, but pTH10 was never lost from cells bearing YEplac112. Thus, SMT4
overexpression cannot suppress a pds5
-null allele, indicating that Smt4p is likely suppressing a defect of the mutant pds5
SMT4 is not a general suppressor of mutants defective in cohesion
Next, we determined whether SMT4
specifically suppresses pds5
mutants or if it is a general suppressor of mutants defective in cohesion, chromosome structure, or cell cycle progression. For this purpose, we assayed the effect of high copy SMT4
(plasmid pTH5) on the temperature sensitivity of mutants in cohesin complex subunits (mcd1
, and smc3
), a protein required for cohesin complex loading on chromosomes (scc2
), topoisomerase II (top2
), a condensin complex subunit (smc2
), and two genes important for cell cycle progression through mitosis (pds1
failed to suppress the cohesion, chromosome topology, or mitotic regulatory mutants, even at semi-permissive temperature ( A, 30 or 34°C; unpublished data). SMT4
did weakly suppress smc2-8
, the condensin complex subunit mutant ( A), consistent with a previous report (Strunnikov et al., 2001
). Because Pds5p is required for condensation as well as cohesion, the weakness of smc2-8
suppression may indicate an indirect suppression due to an effect on wild-type Pds5p by SMT4
or a direct effect on the mutant Smc2p. Thus, SMT4
suppression is largely specific for pds5
Figure 3. Specificity of SMT4 suppression. (A) Effect of high copy SMT4 (2μ URA3 vector) on the temperature-sensitive phenotype of mutants defective in chromosome structure or cell cycle progression. Haploids pds5-1 (VG986-5B), pds5-3 (VG988-1C), mcd1-1 (more ...) SMT4
encodes a SUMO isopeptidase (i.e., deconjugase), and as shown in , wild-type Pds5p is sumoylated. It is possible that SMT4
mutants by decreasing SUMO conjugation of mutant Pds5p. Consistent with this idea, a catalytically dead smt4
allele fails to suppress pds5
mutants (unpublished data). Moreover, overexpressing other SUMO pathway genes should affect pds5
mutants in predictable ways. Proteins that promote SUMO deconjugation should suppress pds5
mutants, whereas proteins that promote SUMO conjugation should be toxic. Therefore, we assayed the effect of high copy plasmids bearing either ULP1
(another SUMO isopeptidase; Li and Hochstrasser, 2000
), or NFI1/SIZ2
(a SUMO E3 ligase; Johnson and Gupta, 2001
) on the temperature sensitivity of pds5
mutants ( B). High copy ULP1
exhibited only a weak suppression compared with SMT4.
In contrast, high copy NFI1
exacerbated the temperature sensitivity of pds5-1
cells. Similar effects were observed in pds5-2
mutants (unpublished data). Thus, overexpression of either SUMO isopeptidase suppresses the pds5
temperature sensitivity, whereas overexpression of a SUMO-conjugating enzyme is toxic. These results support the idea that sumoylation of the mutant Pds5p modulates its activity. Ulp1p localizes to the nuclear periphery, whereas Smt4p localizes to the nucleus (Li and Hochstrasser, 2000
). The differences in their ability to suppress may reflect distinct target specificities, localization, or cell cycle control of their activity.
Figure 4. Sumoylation of Pds5p. (A) IP Western blot of Pds5p from asynchronous cells. PDS5-6MYC (VG2066-7B), PDS5-12MYC (VG2067-2B), and PDS5 (VG982-6A) haploids were grown in YEPD at 23°C, and total protein extracts were made. Pds5p was immunoprecipitated (more ...)
Pds5p is sumoylated in a cell cycle–dependent manner
Given the ability of SUMO pathway genes to either suppress or exacerbate the temperature sensitivity of pds5 mutants, it seemed likely that Pds5p is modified by SUMO. Sumoylation could alter Pds5p function, and the mutant Pds5p might be especially sensitive to this modification. Alternatively, the effect could reflect an indirect effect of SUMO conjugation to another protein. Therefore, we examined whether wild-type Pds5p is sumoylated. Protein extracts from haploid yeast strains bearing MYC epitope–tagged Pds5p (PDS5-6MYC or PDS5-12MYC) or untagged Pds5p (PDS5) were isolated. MYC-tagged Pds5p was immunoprecipitated using anti-MYC antibodies, and then SUMO conjugation was detected by Western blot (see Materials and methods). Two prominent SUMO cross-reacting bands and a third weaker band of higher mobility can be seen in immunoprecipitations (IPs) from PDS5-6MYC and PDS5-12MYC strains ( A, left). In contrast, no SUMO bands were detected in the IP from an untagged strain. The sumoylated forms of Pds5p-6MYC had a mol wt ~20 kD smaller than those for Pds5p-12MYC. This correlates well with the size difference between Pds5p-6MYC and Pds5p-12MYC as shown by Western blot using anti-MYC antibodies ( A, right; see Materials and methods). Because these strains are identical except for the number of MYC tags on Pds5p, these results confirm that Pds5p is sumoylated. As SUMO is added as monomers to target lysines, it appears that three SUMO molecules can be added to Pds5p.
Next, we asked whether Pds5p sumoylation is cell cycle regulated by examining a synchronous population of wild-type (PDS5-6MYC) haploid cells released from S phase at 23°C into YEPD containing α-factor (see Materials and methods). Every 15 min, cell aliquots were processed for isolation of total protein and FACS® analysis. Pds5p SUMO conjugation was detected by MYC IP and Western blot (see Materials and methods). Pds5p sumoylation was low in HU-arrested cells (t = 0), then increased after release from arrest ( B, top). Increased SUMO conjugation was seen at 15 min, which is before bulk DNA replication (). Replication was detected at 30 min and completed at 45 min ( D). It was difficult to ascertain when Pds5p sumoylation achieves a maximum because Pds5p levels decrease after release from HU arrest ( B, bottom; C). Therefore, we performed densitometry to calculate the SUMO/Pds5p ratio. To plot relative sumoylation, we compared the SUMO/Pds5p ratio for HU-arrested cells to that from each time point after release. The ratio was set at one for HU-arrested cells ( E). SUMO conjugation increased on release, and reached a maximum at 75 min. Cells in G2 phase and M phase before anaphase have a single round DNA mass, whereas those in anaphase or telophase have an elongated DNA mass or two separated DNA masses, respectively. We scored DNA morphology and found no evidence for anaphase onset through 60 min, but by 75 min, 20% of cells are in anaphase or telophase. By 105 min, 64% are anaphase or telophase, and 9% have reached G1 phase (unpublished data). Thus, Pds5p SUMO conjugation begins before DNA replication and reaches its maximum at 75 min, when anaphase onset is first detected ( E).
Finally, we assessed Pds5p sumoylation in cells arrested in either G1 phase (α-factor), S phase (HU), metaphase (Nz), and telophase (cdc15) as described for F. As before, densitometry of the SUMO/Pds5p ratio was normalized to HU-arrested cells. Pds5p sumoylation was similar in HU- and Nz-arrested cells, but was almost absent in telophase or G1 cells ( G). Thus, in cycling cells, Pds5p sumoylation increases from S phase through mitosis, but in cells arrested in S phase or metaphase, sumoylation is low. These results indicate that Pds5p sumoylation increases when chromosomes are undergoing dynamic morphological changes, including cohesion establishment, condensation, and dissolution of cohesion.
Smt4p and Nfi1p regulate the levels of Pds5p sumoylation
SMT4 suppresses the temperature sensitivity of pds5 mutants and Pds5p is sumoylated, indicating a connection between Smt4p activity and Pds5p function. Therefore, we assayed whether Smt4p activity modulates Pds5p sumoylation. Wild-type haploid (PDS5-6MYC) strains bearing either SMT4 under control of a galactose-inducible promoter (pGAL-SMT4) or the pGAL promoter alone integrated at TRP1, were grown asynchronously at 23°C in YEP raffinose, and then galactose was added to induce the pGAL promoter (see Materials and methods). Cell aliquots were processed for isolation of total protein and FACS® analysis. Pds5p SUMO conjugation was detected by MYC IP and Western blot (see Materials and methods). Two Pds5p-SUMO bands were seen in raffinose-grown cells ( A, top). In contrast, 1 h after galactose addition, sumoylated Pds5p was absent in the strain bearing pGAL-SMT4, but remained in the pGAL-bearing strain. Total Pds5p levels were unaffected by induction of pGAL-SMT4 ( A, bottom). Thus, SMT4 overexpression rapidly decreases Pds5p sumoylation.
Figure 5. Modulation of Pds5p sumoylation by SUMO pathway genes. Pds5p sumoylation determined by IP Western blot as described in A. (A) Effect of SMT4 overexpression. Haploids containing pGAL-SMT4 (VG2525-2B) or pGAL (VG2524-1A) were grown in YEP raffinose (more ...)
Next, we assayed Pds5p sumoylation in smt4 mutants. Wild-type and smt4 mutant strains bearing Pds5p-6MYC were grown asynchronously in YEPD at 23°C, and were then incubated for 3 h at 37°C, the nonpermissive temperature for the smt4 mutant. Aliquots of cells from 23 and 37°C were processed to assess Pds5p sumoylation and FACS® analysis (see Materials and methods). Pds5p sumoylation was greatly increased in smt4 mutants ( B). Finally, we compared wild-type (Pds5p-6MYC) cells containing high copy plasmids bearing NFI1, SMT4, or ULP1. Overexpression of NFI1 greatly increased Pds5p sumoylation and SMT4 nearly eliminated it, whereas ULP1 moderately decreased sumoylation ( C). These results indicate that Pds5p sumoylation is regulated mainly by Nfi1p and Smt4p activity, and is consistent with the idea that sumoylation modulates Pds5p function.
Overexpression of SMT4 during a single cell cycle window suppresses the inviability and precocious sister dissociation of pds5 mutants
Continuous SMT4 overexpression suppressed pds5 mutants. To map the cell cycle stage where suppression occurs, we overexpressed SMT4 during a single cell cycle window from S phase through mitosis. pds5-2 cells containing PDS1-3HA and either pGAL-SMT4 or pGAL alone were arrested in S phase at 23°C in YEP raffinose. Galactose was added to induce the pGAL promoter (30 min), cells were incubated at 37°C (30 min), and were then released from S phase into fresh 37°C YEP raffinose + galactose containing α-factor (see Materials and methods). By this regimen, the cohesin complex and mutant Pds5-2p are presumed to load normally onto chromosomes in early S phase at permissive temperature, and then SMT4 is overexpressed at the time when a small fraction of Pds5p is sumoylated (). Incubation at 37°C inactivates mutant Pds5-2p, and release at 37°C enables cells to progress through mitosis until rearrest in G1 phase with SMT4 overexpressed at the nonpermissive temperature. Cell aliquots were plated for viability or fixed to monitor cell cycle progression (FACS®, cell morphology, and Pds1p levels) and sister chromatid cohesion (see Materials and methods).
For pds5-2 cells with pGAL alone, viability decreased between 45 and 75 min after release ( A). SMT4 overexpression largely suppressed this decreased viability ( A). DNA replication was completed by 30 min in pds5 cells, regardless of SMT4 overexpression, and in wild-type cells ( B), revealing that the decreased viability of pds5-2 cells with pGAL alone occurs after replication. One possible explanation for this suppression was that SMT4 overexpression abrogates the cell cycle delay of pds5 mutants at 37°C (). However, FACS® profiles and Pds1p Western blots showed that pds5 cells overexpressing SMT4 still delayed in mitosis as compared with wild-type cells (). To assess when anaphase onset normally occurs, we scored DNA morphology in wild-type cells by counting anaphase (stretched DNA) or telophase (two separated DNA masses) cells. At 30 min, few anaphase cells were detected, but by 45 min, 25% of cells were in anaphase or telophase, and such cells increased to 60% by 60 min ( D). Thus, pds5 cells lose viability around the time of anaphase onset, and SMT4 overexpression prevents this viability decrease.
Figure 6. Effect of SMT4 overexpression on viability and cell cycle progression of pds5 cells. Mutant pds5-2 cells bearing either pGAL-SMT4 (VG2445-5B) or pGAL (VG2446-6A) and wild-type cells (VG2452-7A) were grown in YEP raffinose at 23°C, were arrested (more ...)
We assayed sister separation in pds5 cells and by 30 min, few cells with two GFP signals are detected, indicating that cohesion exists regardless of SMT4 expression ( A). By 45 min, separated sisters were seen in 36% of pGAL cells, but in only 23% of pGAL-SMT4 cells. By 60 min, separated sisters were detected in 62% of pGAL cells, but in only 38% of pGAL-SMT4 cells. Separated sisters remained close together in pds5 cells regardless of SMT4 expression ( B). In contrast, in wild-type cells, separated sisters are far apart due to normal anaphase chromosome segregation. Pds1p levels remain high in pds5 cells with pGAL-SMT4, so there must still be some precocious dissociation of one or more chromosomes, which activates the spindle damage checkpoint and inhibits anaphase spindle elongation. The most simple explanation is that SMT4 overexpression prevents cell inviability in pds5 mutants by reducing precocious sister dissociation during mitosis.
Figure 7. SMT4 overexpression suppresses precocious sister chromatid dissociation in pds5 cells. Mutant pds5-2 cells bearing either pGAL-SMT4 (VG2445-5B) or pGAL (VG2446-6A) grown as described in were fixed to monitor cohesion near URA3. (A) Percentage of (more ...)