Pds5p regulates the maintenance of sister chromatid cohesion and is sumoylated to promote the dissolution of cohesion

Pds5p and the cohesin complex are required for sister chromatid cohesion and localize to the same chromosomal loci over the same cell cycle window. However, Pds5p and the cohesin complex likely have distinct roles in cohesion. We report that pds5 mutants establish cohesion, but during mitosis exhibit precocious sister dissociation. Thus, unlike the cohesin complex, which is required for cohesion establishment and maintenance, Pds5p is required only for maintenance. We identified SMT4, which encodes a SUMO isopeptidase, as a high copy suppressor of both the temperature sensitivity and precocious sister dissociation of pds5 mutants. In contrast, SMT4 does not suppress temperature sensitivity of cohesin complex mutants. Pds5p is SUMO conjugated, with sumoylation peaking during mitosis. SMT4 overexpression reduces Pds5p sumoylation, whereas smt4 mutants have increased Pds5p sumoylation. smt4 mutants were previously shown to be defective in cohesion maintenance during mitosis. These data provide the first link between a protein required for cohesion, Pds5p, and sumoylation, and suggest that Pds5p sumoylation promotes the dissolution of cohesion.     

The Ulp2 SUMO protease is required for cell division following termination of the DNA damage checkpoint

Eukaryotic genome integrity is maintained via a DNA damage checkpoint that recognizes DNA damage and halts the cell cycle at metaphase, allowing time for repair. Checkpoint signaling is eventually terminated so that the cell cycle can resume. How cells restart cell division following checkpoint termination is poorly understood. Here we show that the SUMO protease Ulp2 is required for resumption of cell division following DNA damage-induced arrest in Saccharomyces cerevisiae, although it is not required for DNA double-strand break repair. The Rad53 branch of the checkpoint pathway generates a signal countered by Ulp2 activity following DNA damage. Interestingly, unlike previously characterized adaptation mutants, ulp2Delta mutants do not show persistent Rad53 phosphorylation following DNA damage, suggesting checkpoint signaling has been terminated and no longer asserts an arrest in these cells. Using Cdc14 localization as a cell cycle indicator, we show that nearly half of cells lacking Ulp2 can escape a checkpoint-induced metaphase arrest despite their inability to divide again. Moreover, half of permanently arrested ulp2Delta cells show evidence of an aberrant mitotic spindle, suggesting that Ulp2 is required for proper spindle dynamics during cell cycle resumption following a DNA damage-induced cell cycle arrest.     

The SUMO isopeptidase Ulp2p is required to prevent recombination-induced chromosome segregation lethality following DNA replication stress

SUMO conjugation is a key regulator of the cellular response to DNA replication stress, acting in part to control recombination at stalled DNA replication forks. Here we examine recombination-related phenotypes in yeast mutants defective for the SUMO de-conjugating/chain-editing enzyme Ulp2p. We find that spontaneous recombination is elevated in ulp2 strains and that recombination DNA repair is essential for ulp2 survival. In contrast to other SUMO pathway mutants, however, the frequency of spontaneous chromosome rearrangements is markedly reduced in ulp2 strains, and some types of rearrangements arising through recombination can apparently not be tolerated. In investigating the basis for this, we find DNA repair foci do not disassemble in ulp2 cells during recovery from the replication fork-blocking drug methyl methanesulfonate (MMS), corresponding with an accumulation of X-shaped recombination intermediates. ulp2 cells satisfy the DNA damage checkpoint during MMS recovery and commit to chromosome segregation with similar kinetics to wild-type cells. However, sister chromatids fail to disjoin, resulting in abortive chromosome segregation and cell lethality. This chromosome segregation defect can be rescued by overproducing the anti-recombinase Srs2p, indicating that recombination plays an underlying causal role in blocking chromatid separation. Overall, our results are consistent with a role for Ulp2p in preventing the formation of DNA lesions that must be repaired through recombination. At the same time, Ulp2p is also required to either suppress or resolve recombination-induced attachments between sister chromatids. These opposing defects may synergize to greatly increase the toxicity of DNA replication stress.     

The role of karyopherins in the regulated sumoylation of septins

In the yeast Saccharomyces cerevisiae, several components of the septin ring are sumoylated during anaphase and then abruptly desumoylated at cytokinesis. We show that septin sumoylation is controlled by the interactions of two enzymes of the sumoylation pathway, Siz1p and Ulp1p, with the nuclear transport machinery. The E3 ligase Siz1p is imported into the nucleus by the karyopherin Kap95p during interphase. In M phase, Siz1p is exported from the nucleus by the karyopherin Kap142p/Msn5p and subsequently targeted to the septin ring, where it participates in septin sumoylation. We also show that the accumulation of sumoylated septins during mitosis is dependent on the interactions of the SUMO isopeptidase Ulp1p with Kap121p and Kap95p-Kap60p and the nuclear pore complex (NPC). In addition to sequestering Ulp1 at the NPC, Kap121p is required for targeting Ulp1p to the septin ring during mitosis. We present a model in which Ulp1p is maintained at the NPC during interphase and transiently interacts with the septin ring during mitosis.     

Pli1PIAS1 SUMO Ligase Protected by the Nuclear Pore-associated SUMO Protease Ulp1SENP1/2

Covalent modification of the proteome by SUMO is critical for genetic stability and cell growth. Equally crucial to these processes is the removal of SUMO from its targets by the Ulp1 (HuSENP1/2) family of SUMO proteases. Ulp1 activity is normally spatially restricted, because it is localized to the nuclear periphery via interactions with the nuclear pore. Delocalization of Ulp1 causes DNA damage and cell cycle defects, phenotypes thought to be caused by inappropriate desumoylation of nucleoplasmic targets that are normally spatially protected from Ulp1. Here, we define a novel consequence of Ulp1 deregulation, with a major impact on SUMO pathway function. In fission yeast lacking Nup132 (Sc/HuNUP133), Ulp1 is delocalized and can no longer antagonize sumoylation of the PIAS family SUMO E3 ligase, Pli1. Consequently, SUMO chain-modified Pli1 is targeted for proteasomal degradation by the concerted action of a SUMO-targeted ubiquitin ligase (STUbL) and Cdc48-Ufd1-Npl4. Pli1 degradation causes the profound SUMO pathway defects and associated centromere dysfunction in cells lacking Nup132. Thus, perhaps counterintuitively, Ulp1-mediated desumoylation can promote SUMO modification by stabilizing a SUMO E3 ligase.     

Noncovalent binding of small ubiquitin-related modifier (SUMO) protease to SUMO is necessary for enzymatic activities and cell growth

SUMO proteases possess two enzymatic activities to hydrolyze the C-terminal region of SUMOs (hydrolase activity) and to remove SUMO from SUMO-conjugated substrates (isopeptidase activity). SUMO proteases bind to SUMOs noncovalently, but the physiological roles of the binding in the functions of SUMO proteases are not well understood. In this study we found that SUMO proteases (Axam, SENP1, and yeast Ulp1) show different preferences for noncovalent binding to various SUMOs (SUMO-1, -2, -3, and yeast Smt3) and that the hydrolase and isopeptidase activities of SUMO proteases are dependent on their binding to SUMOs through salt bridge. Expression of Smt3 suppressed the phenotype of yeast mutant lacking smt3, which exhibits growth arrest, and the binding of Ulp1 to Smt3 was essential for this rescue activity. Although expression of an Smt3 mutant (smt3R64E(GG)), which conjugates to substrate but loses the ability to bind to Ulp1, rescued the phenotype of yeast lacking smt3 partially, the mutant cells showed an increment in the doubling time and a delay of desumoylation of Smt3-conjugated Cdc3. These results indicate that the noncovalent binding of SUMO protease to SUMO through salt bridge is essential for the enzymatic activities and that the balance between sumoylation and desumoylation is important for cell growth control.     

Smt3/SUMO and Ubc9 are required for efficient APC/C-mediated proteolysis in budding yeast

Ubiquitin-mediated proteolysis triggered by the anaphase-promoting complex/cyclosome (APC/C) is essential for sister chromatid separation and the mitotic exit. Like ubiquitylation, protein modification with the small ubiquitin-related modifier SUMO appears to be important during mitosis, because yeast cells impaired in the SUMO-conjugating enzyme Ubc9 were found to be blocked in mitosis and defective in cyclin degradation. Here, we analysed the role of SUMOylation in the metaphase/anaphase transition and in APC/C-mediated proteolysis in Saccharomyces cerevisiae. We show that cells depleted of Ubc9 or Smt3, the yeast SUMO protein, mostly arrested with undivided nuclei and with high levels of securin Pds1. This metaphase block was partially relieved by a deletion of PDS1. The absence of Ubc9 or Smt3 also resulted in defects in chromosome segregation. Temperature-sensitive ubc9-2 mutants were delayed in proteolysis of Pds1 and of cyclin Clb2 during mitosis. The requirement of SUMOylation for APC/C-mediated degradation was tested more directly in G1-arrested cells. Both ubc9-2 and smt3-331 mutants were defective in efficient degradation of Pds1 and mitotic cyclins, whereas proteolysis of unstable proteins that are not APC/C substrates was unaffected. We conclude that SUMOylation is needed for efficient proteolysis mediated by APC/C in budding yeast.     

SUMO conjugation and deconjugation

Ligation of the ubiquitin-like protein SUMO (Smt3p) to other proteins is essential for viability of the yeast Saccharomyces cerevisiae. Like ubiquitin (Ub), SUMO undergoes ATP-dependent activation by a specific activating enzyme. SUMO-activating enzyme is a heterodimer composed of Uba2p and Aos1p, polypeptides with sequence similarities, respectively, to the C- and N-terminal parts of Ub-activating enzyme. To study the function of SUMO conjugation, we isolated uba2 mutants that were temperature-sensitive for growth. In these mutants conjugation of SUMO to other proteins was drastically reduced, even at the temperature permissive for growth. In a screen for spontaneous suppressors of the temperature-sensitive growth phenotype of the mutant uba2-ts9, we isolated a strain with a null mutation (sut9) in a gene of hitherto unknown function (SUT9/YIL031W/SMT4). This gene encodes a protein with similarities to Ulp1p, a dual-function protease that processes the SUMO precursor and deconjugates SUMO from its substrates. The novel protein was therefore termed Ulp2p. Inactivation of ULP2 in a strain expressing wild-type SUMO-activating enzyme resulted in slow and temperature-sensitive growth, and accumulation of SUMO conjugates. Thus, mutations in SUMO-activating enzyme and mutations in Ulp2p suppress each other, indicating that SUMO conjugation and deconjugation must be in balance for cells to grow normally. Other phenotypes of ulp2 mutants include a defect in cell cycle progression, hypersensitivity to DNA damage, and chromosome mis-segregation. Ulp2p is predominantly located within the nucleus, whereas Ulp1p colocalizes with nuclear pore complex proteins, indicating that the apparently distinct functions of the two SUMO deconjugating enzymes are spatially separated. Received: 1 March 2000 / Accepted: 22 March 2000     

Topoisomerase I-dependent viability loss in saccharomyces cerevisiae mutants defective in both SUMO conjugation and DNA repair

Siz1 and Siz2/Nfi1 are the two Siz/PIAS SUMO E3 ligases in Saccharomyces cerevisiae. Here we show that siz1Delta siz2Delta mutants fail to grow in the absence of the homologous recombination pathway or the Fen1 ortholog RAD27. Remarkably, the growth defects of mutants such as siz1Delta siz2Delta rad52Delta are suppressed by mutations in TOP1, suggesting that these growth defects are caused by topoisomerase I activity. Other mutants that affect SUMO conjugation, including a ulp1 mutant and the nuclear pore mutants nup60Delta and nup133Delta, show similar top1-suppressible synthetic defects with DNA repair mutants, suggesting that these phenotypes also result from reduced SUMO conjugation. siz1Delta siz2Delta mutants also display TOP1-independent genome instability phenotypes, including increased mitotic recombination and elongated telomeres. We also show that SUMO conjugation, TOP1, and RAD27 have overlapping roles in telomere maintenance. Top1 is sumoylated, but Top1 does not appear to be the SUMO substrate involved in the synthetic growth defects. However, sumoylation of certain substrates, including Top1 itself and Tri1 (YMR233W), is enhanced in the absence of Top1 activity. Sumoylation is also required for growth of top1Delta cells. These results suggest that the SUMO pathway has a complex effect on genome stability that involves several mechanistically distinct processes.     

Chromosome Separation and Exit from Mitosis in Budding Yeast: Dependence on Growth Revealed by cAMP-Mediated Inhibition

Cell cycle progression of somatic cells depends on net mass accumulation. In Saccharomyces cerevisiae the cAMP-dependent kinases (PKAs) promote cytoplasmic growth and modulate the growth-regulated mechanism triggering the begin of DNA synthesis. By altering the cAMP signal in budding yeast cells we show here that mitotic events can also depend on growth. In fact, the hyperactivation of PKAs permanently inhibited both anaphase and exit from mitosis when cell growth was repressed. In S. cerevisiae the anaphase promoting complex (APC) triggers entry into anaphase by mediating the degradation of Pds1p. The cAMP pathway activation was lethal together with a partial impairment of the Cdc16p APC subunit, causing a preanaphase arrest, and conversely low PKA activity suppressed the lethality of cdc16-1 cells. Deregulated PKAs partially prevented the decrease of Pds1p intracellular levels concomitantly with the anaphase inhibition, and the PKA-dependent preanaphase arrest could be suppressed in pds1(-) cells. Thus, the cAMP pathway and APC functionally interact in S. cerevisiae and Pds1p is required for the cAMP-mediated inhibition of chromosome separation. Exit from mitosis requires APC, Cdc15p, and the polo-like Cdc5p kinase. PKA hyperactivation and a cdc15 mutation were synthetically lethal and brought to a telophase arrest. Finally, a low cAMP signal allowed cell division at a small cell size and suppressed the lethality of cdc15-2 or cdc5-1 cells. We propose that mitosis progression and the M/G1 phase transition specifically depend on cell growth through a mechanism modulated by PKAs and interacting with the APC/CDC15/CDC5 mitotic system. A possible functional antagonism between PKAs and the mitosis promoting factor is also discussed.     

The Polo-like kinase Cdc5p and the WD-repeat protein Cdc20p/fizzy are regulators and substrates of the anaphase promoting complex in Saccharomyces cerevisiae.

Proteolysis mediated by the anaphase promoting complex (APC) has a crucial role in regulating the passage of cells through anaphase. Destruction of the anaphase inhibitor Pds1p is necessary for separation of sister chromatids, whereas destruction of the mitotic cyclin Clb2p is important for disassembly of the mitotic spindle, cytokinesis and re-replication of the genome. Pds1p proteolysis precedes that of Clb2p by at least 15 min, which helps to ensure that cells never re-replicate their genome before they have separated sister chromatids at the previous mitosis. What triggers Pds1p proteolysis and why does it not also trigger that of Clb2p? Apart from sharing a dependence on the APC, these two proteolytic events differ in their dependence on other cofactors. Pds1p proteolysis depends on a WD-repeat protein called Cdc20p, whereas Clb2p proteolysis depends on another, related WD protein called Hct1/Cdh1p. On the other hand, destruction of Clb2p, but not that of Pds1p, depends on the Polo-like kinase, Cdc5p. Cdc20p is essential for separation of sister chromatids, whereas Cdc5p is not. We show that both Cdc5p and Cdc20p are unstable proteins whose proteolysis is regulated by the APC. Both proteins accumulate during late G2/M phase and disappear at a late stage of anaphase. Accumulation of Cdc20p contributes to activation of Pds1p proteolysis in metaphase, whereas accumulation of Cdc5p facilitates the activation of Clb2p proteolysis.     

Pds1p, an inhibitor of anaphase in budding yeast, plays a critical role in the APC and checkpoint pathway(s)

We report the isolation and characterization of pds1 mutants in Saccharomyces cerevisiae. The initial pds1-1 allele was identified by its inviability after transient exposure to microtubule inhibitors and its precocious dissociation of sister chromatids in the presence of these microtubule inhibitors. These findings suggest that pds1 mutants might be defective in anaphase arrest that normally is imposed by a spindle-damage checkpoint. To further examine a role for Pds1p in anaphase arrest, we compared the cell cycle arrest of pds1 mutants and PDS1 cells after: (a) the inactivation of Cdc16p or Cdc23p, two proteins that are required for the degradation of mitotic cyclins and are putative components of the yeast anaphase promoting complex (APC); (b) the inactivation of Cdc20p, another protein implicated in the degradation of mitotic cyclins; and (c) the inactivation of Cdc13 protein or gamma irradiation, two circumstances that induce a DNA- damage checkpoint. Under all these conditions, anaphase is inhibited in PDS1 cells but not in pds1 mutants. From these results we suggest that Pds1 protein is an anaphase inhibitor in PDS1 cells but not in pds1 mutants. From these results we suggest that Pds1 protein is an anaphase inhibitor that plays a critical role in the control of anaphase by both APC and checkpoints. We also show that pds1 mutants exit mitosis and initiate new rounds of cell division after gamma irradiation and Cdc13p inactivation but no after nocodazole-treatment or inactivation of Cdc16p, Cdc20p or Cdc23p function. Therefore, in the DNA-damage checkpoint, Pds1p is required for the inhibition of cytokinesis and DNA replication as well as anaphase. The role of Pds1 protein in anaphase inhibition and general cell cycle regulation is discussed.     

The Pds1 anaphase inhibitor and Mec1 kinase define distinct checkpoints coupling S phase with mitosis in budding yeast

In most eukaryotic cells, DNA replication is confined to S phase of the cell cycle [1]. During this interval, S-phase checkpoint controls restrain mitosis until replication is complete [2]. In budding yeast, the anaphase inhibitor Pds1p has been associated with the checkpoint arrest of mitosis when DNA is damaged or when mitotic spindles have formed aberrantly [3] [4], but not when DNA replication is blocked with hydroxyurea (HU). Previous studies have implicated the protein kinase Mec1p in S-phase checkpoint control [5]. Unlike mec1 mutants, pds1 mutants efficiently inhibit anaphase when replication is blocked. This does not, however, exclude an essential S-phase checkpoint function of Pds1 beyond the early S-phase arrest point of a HU block. Here, we show that Pds1p is an essential component of a previously unsuspected checkpoint control system that couples the completion of S phase with mitosis. Further, the S-phase checkpoint comprises at least two distinct pathways. A Mec1p-dependent pathway operates early in S phase, but a Pds1p-dependent pathway becomes essential part way through S phase.     

RanBP2 and SENP3 function in a mitotic SUMO2/3 conjugation-deconjugation cycle on Borealin

The ubiquitin-like SUMO system controls cellular key functions, and several lines of evidence point to a critical role of SUMO for mitotic progression. However, in mammalian cells mitotic substrates of sumoylation and the regulatory components involved are not well defined. Here, we identify Borealin, a component of the chromosomal passenger complex (CPC), as a mitotic target of SUMO. The CPC, which additionally comprises INCENP, Survivin, and Aurora B, regulates key mitotic events, including chromosome congression, the spindle assembly checkpoint, and cytokinesis. We show that Borealin is preferentially modified by SUMO2/3 and demonstrate that the modification is dynamically regulated during mitotic progression, peaking in early mitosis. Intriguingly, the SUMO ligase RanBP2 interacts with the CPC, stimulates SUMO modification of Borealin in vitro, and is required for its modification in vivo. Moreover, the SUMO isopeptidase SENP3 is a specific interaction partner of Borealin and catalyzes the removal of SUMO2/3 from Borealin. These data thus delineate a mitotic SUMO2/3 conjugation–deconjugation cycle of Borealin and further assign a regulatory function of RanBP2 and SENP3 in the mitotic SUMO pathway.     

Cell cycle-regulated attachment of the ubiquitin-related protein SUMO to the yeast septins

SUMO is a ubiquitin-related protein that functions as a posttranslational modification on other proteins. SUMO conjugation is essential for viability in Saccharomyces cerevisiae and is required for entry into mitosis. We have found that SUMO is attached to the septins Cdc3, Cdc11, and Shs1/Sep7 specifically during mitosis, with conjugates appearing shortly before anaphase onset and disappearing abruptly at cytokinesis. Septins are components of a belt of 10-nm filaments encircling the yeast bud neck. Intriguingly, only septins on the mother cell side of the bud neck are sumoylated. We have identified four major SUMO attachment-site lysine residues in Cdc3, one in Cdc11, and two in Shs1, all within the consensus sequence (IVL)KX(ED). Mutating these sites eliminated the vast majority of bud neck-associated SUMO, as well as the bulk of total SUMO conjugates in G(2)/M-arrested cells, indicating that sumoylated septins are the most abundant SUMO conjugates at this point in the cell cycle. This mutant has a striking defect in disassembly of septin rings, resulting in accumulation of septin rings marking previous division sites. Thus, SUMO conjugation plays a role in regulating septin ring dynamics during the cell cycle.     

The yeast ULP2 (SMT4) gene encodes a novel protease specific for the ubiquitin-like Smt3 protein

Yeast Smt3 and its vertebrate homolog SUMO-1 are ubiquitin-like proteins (Ubls) that are reversibly ligated to other proteins. Like SMT3, SMT4 was first isolated as a high-copy-number suppressor of a defective centromere-binding protein. We show here that SMT4 encodes an Smt3-deconjugating enzyme, Ulp2. In cells lacking Ulp2, specific Smt3-protein conjugates accumulate, and the conjugate pattern is distinct from that observed in a ulp1(ts) strain, which is defective for a distantly related Smt3-specific protease, Ulp1. The ulp2Delta mutant exhibits a pleiotropic phenotype that includes temperature-sensitive growth, abnormal cell morphology, decreased plasmid and chromosome stability, and a severe sporulation defect. The mutant is also hypersensitive to DNA-damaging agents, hydroxyurea, and benomyl. Although cell cycle checkpoint arrest in response to DNA damage, replication inhibition, or spindle defects occurs with normal kinetics, recovery from arrest is impaired. Surprisingly, either introduction of a ulp1(ts) mutation or overproduction of catalytically inactive Ulp1 can substantially overcome the ulp2Delta defects. Inactivation of Ulp2 also suppresses several ulp1(ts) defects, and the double mutant accumulates far fewer Smt3-protein conjugates than either single mutant. Our data suggest the existence of a feedback mechanism that limits Smt3-protein ligation when Smt3 deconjugation by both Ulp1 and Ulp2 is compromised, allowing a partial recovery of cell function.     

The yeast homologue of the microtubule-associated protein Lis1 interacts with the sumoylation machinery and a SUMO-targeted ubiquitin ligase

Microtubules and microtubule-associated proteins are fundamental for multiple cellular processes, including mitosis and intracellular motility, but the factors that control microtubule-associated proteins (MAPs) are poorly understood. Here we show that two MAPs—the CLIP-170 homologue Bik1p and the Lis1 homologue Pac1p—interact with several proteins in the sumoylation pathway. Bik1p and Pac1p interact with Smt3p, the yeast SUMO; Ubc9p, an E2; and Nfi1p, an E3. Bik1p interacts directly with SUMO in vitro, and overexpression of Smt3p and Bik1p results in its in vivo sumoylation. Modified Pac1p is observed when the SUMO protease Ulp1p is inactivated. Both ubiquitin and Smt3p copurify with Pac1p. In contrast to ubiquitination, sumoylation does not directly tag the substrate for degradation. However, SUMO-targeted ubiquitin ligases (STUbLs) can recognize a sumoylated substrate and promote its degradation via ubiquitination and the proteasome. Both Pac1p and Bik1p interact with the STUbL Nis1p-Ris1p and the protease Wss1p. Strains deleted for RIS1 or WSS1 accumulate Pac1p conjugates. This suggests a novel model in which the abundance of these MAPs may be regulated via STUbLs. Pac1p modification is also altered by Kar9p and the dynein regulator She1p. This work has implications for the regulation of dynein''s interaction with various cargoes, including its off-loading to the cortex.     

Mitotic Cdc6 stabilizes anaphase-promoting complex substrates by a partially Cdc28-independent mechanism, and this stabilization is suppressed by deletion of Cdc55

Ectopic expression of Cdc6p results in mitotic delay, and this has been attributed to Cdc6p-mediated inhibition of Cdc28 protein kinase and failure to activate the anaphase-promoting complex (APC). Here we show that endogenous Cdc6p delays a specific subset of mitotic events and that Cdc28 inhibition is not sufficient to account for it. The depletion of Cdc6p in G(2)/M cells reveals that Cdc6p is rate limiting for the degradation of the APC/Cdc20 substrates Pds1p and Clb2p. Conversely, the premature expression of Cdc6p delays the degradation of APC/Cdc20 substrates. Abolishing Cdc6p/Cdc28p interaction does not eliminate the Cdc6-dependent delay of these anaphase events. To identify additional Cdc6-mediated, APC-inhibitory mechanisms, we looked for mutants that reversed the mitotic delay. The deletion of SWE1, RAD24, MAD2, or BUB2 had no effect. However, disrupting CDC55, a PP2A regulatory subunit, suppressed the Cdc6p-dependent delay of Pds1 and Clb2 destruction. A specific role for CDC55 was supported by demonstrating that the lethality of Cdc6 ectopic expression in a cdc16-264 mutant is suppressed by the deletion of CDC55, that endogenous Cdc6p coimmunoprecipitates with the Cdc55 and Tpd3 subunits of PP2A, that Cdc6p/Cdc55p/Tpd3 interaction occurs only during mitosis, and that Cdc6 affects PP2A-Cdc55 activity during anaphase. This demonstrates that the levels and timing of accumulation of Cdc6p in mitosis are appropriate for mediating the modulation of APC/Cdc20.     

The SUMO isopeptidase Ulp2 prevents accumulation of SUMO chains in yeast

The ubiquitin-related protein SUMO functions by becoming covalently attached to lysine residues in other proteins. Unlike ubiquitin, which is often linked to its substrates as a polyubiquitin chain, only one SUMO moiety is attached per modified site in most substrates. However, SUMO has recently been shown to form chains in vitro and in mammalian cells, with a lysine in the non-ubiquitin-like N-terminal extension serving as the major SUMO-SUMO branch site. To investigate the physiological function of SUMO chains, we generated Saccharomyces cerevisiae strains that expressed mutant SUMOs lacking various lysine residues. Otherwise wild-type strains lacking any of the nine lysines in SUMO were viable, had no obvious growth defects or stress sensitivities, and had SUMO conjugate patterns that did not differ dramatically from wild type. However, mutants lacking the SUMO-specific isopeptidase Ulp2 accumulated high molecular weight SUMO-containing species, which formed only when the N-terminal lysines of SUMO were present, suggesting that they contained SUMO chains. Furthermore SUMO branch-site mutants suppressed several of the phenotypes of ulp2delta, consistent with the possibility that some ulp2delta phenotypes are caused by accumulation of SUMO chains. We also found that a mutant SUMO whose non-ubiquitin-like N-terminal domain had been entirely deleted still carried out all the essential functions of SUMO. Thus, the ubiquitin-like domain of SUMO is sufficient for conjugation and all downstream functions required for yeast viability. Our data suggest that SUMO can form chains in vivo in yeast but demonstrate conclusively that chain formation is not required for the essential functions of SUMO in S. cerevisiae.