Pli1^PIAS1 SUMO Ligase Protected by the Nuclear Pore-associated SUMO Protease Ulp1^SENP1/2

Background: SUMO conjugation to the proteome critically controls cell growth, but mechanisms for regulating SUMO ligases are poorly defined.

Results: Desumoylation of the major fission yeast SUMO ligase by a nuclear pore-associated protease protects it from proteolysis.

Conclusion: Desumoylation of a SUMO ligase antagonizes autoinhibition of the SUMO pathway.

Significance: These data demonstrate cooperation between STUbL and Cdc48(p97) in proteasome-mediated degradation of SUMO conjugates.

Abstract

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.

Introduction

Covalent modification of the proteome by the small ubiquitin-like modifier SUMO² regulates most aspects of cell growth, including cell cycle transitions and genome stability (1–4). The addition of SUMO and its removal from target proteins must be tightly regulated to avoid the pathological consequences of defects in pathway homeostasis (5–7).

SUMO is attached to lysine residues in target proteins via an enzymatic cascade of E1 activating, E2 conjugating, and E3 ligase factors (4). E3 ligases provide target specificity and enhance SUMO conjugation. A major family of SUMO E3 ligases is the PIAS (protein inhibitor of activated STAT) family, which has several members in mammalian cells e.g. PIAS1–4, two in budding yeast SIZ1/2, and one called Pli1 in fission yeast (8, 9). Pli1 catalyzes the majority of sumoylation (>90%) including SUMO chain formation, and its deletion causes meiotic defects, centromeric heterochromatin dysfunction, and telomere elongation (8, 10).

SUMO is removed by one of a small family of Ubl-specific proteases (11, 12). In yeasts there are two Ubl-specific proteases, Ulp1 and Ulp2, of which Ulp1 processes SUMO into a mature form by removing a C-terminal peptide to reveal a diglycine motif. Both Ulp1 and Ulp2 desumoylate a subset of SUMO conjugates, with specificity likely driven in large part by their spatial separation (12). Ulp1 localizes to the nuclear rim by associating with nuclear pores, whereas Ulp2 is nucleoplasmic (12–18). In higher eukaryotes, SENP1/2 localize to nuclear pores like Ulp1 (19–21), and SENP6/7, like Ulp2, are nucleoplasmic (22, 23).

Sumoylated proteins can also be ubiquitinated by a SUMO-targeted ubiquitin ligase (STUbL) to promote their degradation at the proteasome (24–26). Correlative evidence suggests that SUMO chains act as targeting signals for STUbLs. Consistent with this, high molecular weight SUMO chains accumulate in STUbL mutant cells (24–26), a phenotype also caused by Ulp2 inactivation (10, 27). Moreover, in fission yeast, the growth and genome stability defects caused by both STUbL and Ulp2 inactivation are suppressed by blocking SUMO chain formation (10, 28). In contrast, preventing SUMO chain formation in budding yeast is lethal to STUbL mutants but suppresses some ulp2Δ defects (27, 29, 30). These findings highlight the importance of SUMO pathway homeostasis and that the “wiring” of the STUbL-SUMO interface differs notably between these yeasts (see above and Refs. 10 and 27–30).

The nuclear pore has emerged as a broadly conserved hub of SUMO-mediated signaling, impacting key processes such as transcription, chromosome segregation, and DNA repair (31, 32). However, despite functional overlap, mutants of orthologous nuclear pore components cause different phenotypes in fission and budding yeast, the latter of which has been used in most studies (33, 34). Therefore, to robustly model how the nuclear pore could impact SUMO pathway homeostasis in higher eukaryotes, it is important to determine how the nuclear pore impacts sumoylation in fission yeast.

Herein we reveal that sumoylation is globally reduced in fission yeast deleted for Nup132, which contrasts with the relatively mild and selective sumoylation defects of analogous budding yeast mutants (16, 35, 36). Nevertheless, we show that as in budding yeast (36), Ulp1 is both delocalized and less abundant in nup132Δ (Sc nup133Δ) cells. What then causes the profound SUMO conjugation defect in nup132Δ cells? Intriguingly, we found that Ulp1 dysfunction in nup132Δ cells allows an accumulation of SUMO chains on the major SUMO E3 ligase Pli1, which promote its degradation in a STUbL-, Cdc48-Ufd1-Npl4-, and proteasome-dependent manner. Moreover, this novel phenomenon allowed us to execute a detailed mechanistic dissection of SUMO pathway and cofactor requirements in the turnover of a specific STUbL substrate. Overall, our data define how the activity of a PIAS family SUMO ligase, and thus its physiological impact, e.g. centromere function, hang in the balance between its autosumoylation and desumoylation by a nuclear pore localized SUMO protease.

Experimental Procedures

Yeast Strains and Growth Conditions

Standard yeast methods were performed as described previously (37). The strains used in this study are listed in Table 1.

TABLE 1.

List of yeast strains used in this study

Strain	Genotypea	Source
NBY752	imr1R (dg-glu)NcoI::ura4 oriI ade6-210/ade6-216	Ref. 51
NBY5450	nup132::ura4+	Ref. 33
NBY780	h+
NBY781	h−
NBY1493	pli1::kanMx6
NBY1820	pli1-TAP:kanMx6
NBY1967	pli1::hphMx6 imr1R (dg-glu)NcoI::ura4 oriI ade6-210/ade6-216
NBY2471	slx8-29:kanMx6
NBY2754	pREP2:ura4+ integrated at ars1
NBY2756	slx8-29:kanMx6 pREP2:ura4+ integrated at ars1
NBY2927	ulp2::kanMx6
NBY3063	ulp1-GFP:kanMx6
NBY3172	ulp2::hphMx6
NBY5159	ulp1-myc13:kanMx6	Ref. 14
NBY5428	nup132::ura4+ slx8-29:kanMx6
NBY5467	nup132::ura4+ ulp2::kanMx6
NBY5468	nup132::ura4+ ulp1-GFP:kanMx6
NBY5471	pREP41-SUMOGG:LEU2 pREP2:ura4+ integrated at ars1
NBY5472	pREP41-SUMOFL:LEU2 pREP2:ura4+ integrated at ars1
NBY5473	slx8-29:kanMx6 pREP41-SUMOGG:LEU2 pREP2:ura4+ integrated at ars1
NBY5474	slx8-29:kanMx6 pREP41-SUMOFL:LEU2 pREP2:ura4+ integrated at ars1
NBY5475	nup132::ura4+ pREP41-SUMOGG:LEU2
NBY5476	nup132::ura4+ pREP41-SUMOFL:LEU2
NBY5477	nup132::ura4+ slx8-29:kanMx6 pREP41-SUMOGG:LEU2
NBY5478	nup132::ura4+ slx8-29:kanMx6 pREP41-SUMOFL:LEU2
NBY5506	pli1-TAP:kanMx6 nup132::ura4+
NBY5528	ulp1-1:kanMx6
NBY5544	pli1-TAP:kanMx6 nup132::ura4+ pREP41-ulp1:LEU2
NBY5546	pli1-TAP:kanMx6 nup132::ura4+ slx8-29:hphMx6
NBY5547	pli1-TAP:kanMx6 slx8-29:hphMx6
NBY5548	pli1-TAP:kanMx6 nup132::ura4+ ufd1-1:natMx6
NBY5549	pli1-TAP:kanMx6 ufd1-1:natMx6
NBY5550	pli1-TAP:kanMx6 nup132::ura4+ mts3-1
NBY5551	pli1-TAP:kanMx6 mts3-1
NBY5569	pli1-TAP:kanMx6 nup132::ura4+ slx8-29:hphMx6 pREP41-6HIS-SUMO:LEU2
NBY5573	pli1-TAP:kanMx6 ulp1-1:kanMx6
NBY5577	pli1::kanMx6 pREP41-pli1:LEU2
NBY5579	nup132::ura4+ pREP41-pli1:LEU2
NBY5582	pli1-TAP:kanMx6 nup132::ura4+ SUMOK14,K30R
NBY5593	nup132::ura4+ slx8-29:kanMx6 pREP41-pli1:LEU2
NBY5620	nup132::ura4+ imr1R (dg-glu)NcoI::ura4 oriI ade6-210/ade6-216
NBY5621	pli1-TAP:kanMx6 nup132::ura4+ SUMOD81R
NBY5633	pli1::kanMx6 ulp2::hphMx6
NBY5665	ulp1-myc13:kanMx6 nup132:hphMx6

^a All strains are of ura4-D18 leu1-32 background genotype, unless otherwise stated. Double colons represent knockouts; single colons represent tagging. A reference is given for strains not generated in this study.

Spot Assays

The cells were grown at 25 °C to logarithmic phase (A₆₀₀ of 0.6–0.8), spotted on agar plates in 5-fold dilutions from a starting A₆₀₀ of 0.5 and then incubated at 25–35 °C for 3–5 days.

Fluorescence Microscopy

GFP fusion proteins and DAPI staining were imaged in live cells using an Eclipse E800 microscope (Nikon Metrology) with a 100× Plan Differential Interference Contrast H oil immersion objective. The images were captured through an INFINITY 3 charge-coupled device camera using the INFINITY ANALYZE software (Lumenera Corporation).

Western Blotting

Exponentially growing cells (∼15 A₆₀₀ units) were washed with STOP buffer (10 mm EDTA, 50 mm NaF, 150 mm NaCl, 1 mm NaN₃). The pellets were flash-frozen in liquid nitrogen, and the cells were lysed by beating twice at 5.0 m/s for 20 s in a FastPrep-24 instrument (MP Biochemicals) in 200 μl of 20% TCA supplied with 100 ml silica-zirconia beads (BioSpec Products). After bead beating, 400 μl of 5% TCA was added, and the total cell lysate was centrifuged at 16,000 × g for 5 min at 4 °C. The pellet was washed twice with 0.1% TCA. The precipitated proteins were resuspended in 8 m urea, 50 mm Tris, pH 8.5, 150 mm NaCl. Protein was quantitated by measuring absorbance at A₂₈₀, and 60 μg of proteins were resolved on a gradient SDS-PAGE gel and transferred to a nitrocellulose membrane. The membrane was blocked in 1% (w/v) nonfat milk in phosphate-buffered saline solution with 0.1% (v/v) Tween 20, probed with peroxidase anti-peroxidase and then detected using an ECL Dura system (Pierce) or probed with antibodies against α-tubulin, Pmt3 (SUMO) and then an IRDye-conjugated secondary antibody and imaged on an ODYSSEY scanner (Li-Cor).

Nickel-Nitrilotriacetic Acid Pulldown

Proteins from cell pellets were extracted by TCA precipitation as described above, and 20 mg of proteins were incubated with 50 μl of nickel-nitrilotriacetic acid beads (Qiagen) in the presence of 15 mm imidazole at room temperature for 1 h. The beads were then washed three times with 8 m urea, 50 mm Tris, pH 8, 150 mm NaCl, and 20 mm imidazole and eluted with 2× LDS sample loading buffer (Invitrogen) at 70 °C for 10 min. Input, flow through, and one-third of the nickel pulldown were resolved by SDS-PAGE and detected by peroxidase anti-peroxidase Western blotting.

RNA Extraction and RT-Quantitative PCR

Total RNA was extracted from ∼5 A₆₀₀ units of exponentially growing cells. The cells were lysed by bead beating in 0.8 ml of TRIzol (Invitrogen), using the equipment and settings described above. RNA extraction with TRIzol was performed by following the manufacturer's instructions. The extracted RNA was treated with DNase I (New England Biolabs) and cleaned up with the RNeasy Plus minikit (Qiagen). A total of 1 μg of DNase-treated RNA was reverse transcribed using the ProtoScript® II first strand cDNA synthesis kit (New England Biolabs). The completed reactions were diluted 10-fold, and 5 μl of the dilutions was used as the template in 20-μl quantitative PCR mixtures using the SensiFAST SYBR No-ROX kit (Bioline), and the quantitative PCRs were carried out using the Chromo4 system (Bio-Rad).

Plasmid Construction

Unless otherwise stated, cDNAs were cloned by PCR amplification using specific primers and PrimeStar DNA polymerase (Clontech) followed by ligation into the pREP series of yeast shuttle vectors (38). Details regarding plasmid construction are available on request.

Results

Nuclear Pore Mutant Suppresses Phenotypes of STUbL Dysfunction

To assess roles of fission yeast Nup132 in SUMO pathway homeostasis and genome stability, we analyzed genetic interactions between its deletion (nup132Δ) and alleles of STUbL (slx8-29) and Ulp2 (ulp2Δ). As in previous reports, nup132Δ cells grew similarly to wild type and exhibited little to no sensitivity to genotoxins or elevated temperatures (Fig. 1A) (33, 34). These subtle phenotypes are consistent with the fact that protein import/export and RNA export pathways are intact in nup132Δ cells and that Nup132 has an expressed paralog called Nup131 (33, 34).

FIGURE 1.

Nup132 deletion suppresses lethality and accumulation of SUMO conjugates in slx8-29 and ulp2Δ mutants. A, serial dilutions of the indicated strains were spotted onto media with indicated drugs, grown at the indicated temperatures. B, Western blots of sumoylated proteins or tubulin of indicated strains grown at 25 °C to log phase or to mid log phase then shifted to 35 °C for 6 h. The black arrowhead indicates the position of free mature SUMO.

Intriguingly, deletion of Nup132 strongly suppressed the growth defects and genotoxin sensitivity of slx8-29 (Fig. 1A). This was surprising, because combined deletions of the analogous factors in budding yeast are synthetically sick (36). As observed for slx8-29, we found that ulp2Δ phenotypes were also suppressed by deleting Nup132 (Fig. 1A). Because an accumulation of SUMO conjugates, particularly chains, drives slx8-29 and ulp2Δ phenotypes (10, 28, 39), we analyzed global sumoylation in Nup132 deleted cells.

At permissive temperature, hypomorphic slx8-29 cells have increased levels of high molecular weight (HMW) SUMO conjugates as compared with wild type (Fig. 1B) (28). In contrast, both nup132Δ and slx8-29 nup132Δ cells had less HMW SUMO conjugates than wild type (Fig. 1B). Moreover, at restrictive temperature, the strong accumulation of HMW SUMO conjugates in slx8-29 cells was absent in slx8-29 nup132Δ cells (Fig. 1B). There was also a lower level of HMW SUMO conjugates in ulp2Δ nup132Δ double mutant versus ulp2Δ single mutant cells (Fig. 1B). Therefore, for as yet undefined reasons, nup132Δ cells exhibit reduced global sumoylation that correlates with the rescue of STUbL and Ulp2 SUMO protease dysfunction.

Altered Ulp1 Activity in nup132Δ Cells

Ulp1 endogenously tagged with GFP (Ulp1-GFP) gives a weak fluorescence signal but shows that fission yeast Ulp1 exhibits a perinuclear localization in live as well as fixed cells (Fig. 2A) (14). Based on analyses of Ulp1 orthologs in other species, this likely reflects its nuclear pore association (32). Budding yeast Ulp1 is anchored at the nuclear pore via a complex set of interactions between the Ulp1 N terminus and several nuclear pore-associated factors, and both its anchoring and protein stability are disrupted in cells lacking Nup133 (fission yeast Nup132; reviewed in Ref. 32). Similarly, we found that both the perinuclear signal of Ulp1 and the level of full-length protein were reduced in nup132Δ cells (Fig. 2, A and B). Thus, the anchoring mechanism of Ulp1 at the nuclear pore is broadly conserved, but the consequences of Ulp1 delocalization and destabilization for SUMO pathway homeostasis are different.

FIGURE 2.

Cells lacking Nup132 have altered Ulp1 activity. A, live cell imaging of GFP fluorescence of Ulp1-GFP and DAPI staining in wild type (top panels) and nup132Δ cells (bottom panels). In addition to defining the nucleus, live cell staining of fission yeast with DAPI also delineates the cell wall and septum. B, Western blots of Ulp1 endogenously tagged with Myc₁₃ or tubulin of indicated strains. The black arrowhead points to the position of full-length Ulp1-Myc₁₃. The asterisk marks the position of a truncated form of Ulp1-Myc₁₃. C, Western blots of sumoylated proteins or tubulin of indicated strains. Ectopic expression of SUMO_GG or SUMO_FL under the nmt41 promoter was induced by growing cells in the absence of thiamine (B1) for 22 h at 25 °C and then an additional 6 h at 35 °C. The black arrowhead indicates the position of free mature SUMO, and the white arrowhead indicates the position of immature SUMO.

Because Ulp1 functions in SUMO maturation, we next tested whether reduced sumoylation in nup132Δ cells was due to inefficient SUMO maturation. We ectopically expressed either full-length (SUMO_FL) or the mature form (SUMO_GG) of SUMO, which both ran at the size of mature SUMO (Fig. 2C, black arrowhead) in wild type cells, indicating that normal Ulp1 activity can fully process overexpressed full-length SUMO. Ulp1 maturation activity is slightly compromised in nup132Δ cells, because they show a small amount of residual immature full-length SUMO (Fig. 2C, white arrowhead). However, because SUMO conjugation in nup132Δ cells is low and unchanged whether they express full-length or mature SUMO (Fig. 2C), we conclude that reduced sumoylation in nup132Δ cells is not caused by a SUMO maturation defect.

Interestingly, nup132Δ slx8-29 cells exhibited a major increase in SUMO conjugates, to a level exceeding that of slx8-29 single mutant cells (Fig. 2C). Therefore, the inactivation of STUbL, when combined with higher levels of SUMO, restores global sumoylation in nup132Δ cells. Enhanced global sumoylation in nup132Δ slx8-29 as compared with slx8-29 cells is consistent with reduced Ulp1-mediated desumoylation in nup132Δ mutants.

The PIAS Family Ligase Pli1 Is Destabilized in Cells Lacking Nup132

In fission yeast, the sole PIAS family ligase called Pli1 is responsible for generating the majority (>90%) of SUMO conjugates detectable by Western blotting (8, 10, 28, 40). Although Pli1 deletion does not overtly affect cell growth, it strongly suppresses STUbL mutants, disrupts centromere function, and causes telomere elongation (8, 10, 28, 40, 41). Because nup132Δ cells share the pli1Δ phenotypes of global hyposumoylation, elongated telomeres, and suppression of STUbL dysfunction (Fig. 1, A and B) (28, 42, 43), we analyzed Pli1 levels in nup132Δ cells.

The stability of endogenous Pli1 was monitored over time in cells treated with cycloheximide, an inhibitor of protein synthesis. Intriguingly, we found that Pli1 was both less abundant and more rapidly degraded in nup132Δ versus wild type cells (Fig. 3A). Given that STUbL inactivation can restore global sumoylation in nup132Δ cells (Fig. 2C), we tested whether the slx8-29 mutation affected Pli1 stability. Strikingly, whereas Pli1 was only weakly detectable in nup132Δ cells, Pli1 abundance and a series of lower mobility species were dramatically increased in slx8-29 nup132Δ cells (Fig. 3B). Neither Pli1 levels nor its gel migration were affected in slx8-29 single mutant cells (Fig. 3B).

FIGURE 3.

Pli1 SUMO ligase is destabilized in nup132Δ cells. A, wild type or nup132Δ cells grown at 25 °C to mid log phase and then shifted to 35 °C for 4 h. Cycloheximide was added to cell cultures at 200 μg/ml, and cells were harvested at 0, 15, 30, 60, or 120 min after cycloheximide addition and analyzed by Western blotting. The levels of Pli1-TAP proteins (unmodified and all SUMO modified species) were normalized to the levels of tubulin in each sample and quantified with National Institutes of Health ImageJ software. The graph is representative of results from three experiments. B, Western blots of Pli1-TAP or tubulin of indicated strains grown at 25 °C to log phase (top panels) or to mid log phase then shifted to 35 °C for 6 h (bottom panels). Black and white arrowheads mark the positions of unmodified and SUMO modified Pli1-TAP bands, respectively.

We and others recently proposed a cooperative role for STUbL and Cdc48 (p97) AAA⁺ ATPase in the proteasomal degradation of SUMO conjugates, at a global level (44, 45). Consistent with this proposal, mutations in the Cdc48 cofactor Ufd1 (ufd1-1) or in the 19S proteasome regulatory subunit Mts3 (mts3-1) also stabilized Pli1 in nup132Δ cells (Fig. 3B).

Direct Evidence that SUMO Chains Are Required for STUbL Targeting

The role of STUbL in targeting SUMO-conjugated proteins is now well established, and notably, slower migrating forms of Pli1 accumulate in slx8-29 nup132Δ cells (Fig. 3B). To confirm that these species are SUMO-conjugated Pli1, we purified SUMO conjugates under denaturing conditions from slx8-29 nup132Δ cells and probed for Pli1 (Fig. 4A). This approach enriched only the slower migrating Pli1 species and revealed higher order polysumoylation of Pli1 (Fig. 4A, white arrowheads).

FIGURE 4.

Pli1 SUMO ligase is stabilized by Ulp1-mediated desumoylation. A, His₆-SUMO was expressed from the nmt41 promoter in slx8-29 nup132Δ cells. Cells were grown in the absence of B1 for 24 h, and proteins were extracted by TCA and pulled down on nickel-nitrilotriacetic acid (Ni) beads. Input (IN), pulldown (PD), and flow through (FT) were analyzed for the presence of Pli1-TAP by peroxidase anti-peroxidase (PAP) Western blotting. B, Western blots of Pli1-TAP or tubulin of indicated strains grown at 25 °C to log phase. C, cells were grown in the absence of B1 for 24 h to induce the ectopic expression of nmt41-driven Ulp1. In the A–C, black and white arrowheads mark the position of unmodified and modified Pli1-TAP bands. D, the transcript levels of ulp1 and pli1 of cells grown in the absence of B1 for 24 h to induce the ectopic expression of nmt41-driven Ulp1 or under repressed condition (+ B1) were analyzed by RT-quantitative PCR.

STUbLs are thought to target proteins that are modified by SUMO chains, and strong correlative data have been published (see Refs. 25, 26, and 46–48). To test the role of SUMO chains more directly, we used two SUMO mutants that we previously showed block SUMO chain formation in different ways (10).

SUMO chains form through Lys¹⁴ and Lys³⁰ in fission yeast SUMO and are therefore absent in SUMO^K14,30R mutant cells (10, 49). Strikingly, Pli1 was stabilized in nup132Δ SUMO^K14,30R cells (Fig. 4B), to an extent similar to that seen in nup132Δ slx8-29 cells (Fig. 3B). Moreover, the SUMO^D81R mutation, which abolishes the noncovalent SUMO:Ubc9 complex required for SUMO chain formation (10), also stabilized Pli1 in nup132Δ cells (Fig. 4B).

In light of the above data, it is interesting to note that the absence of SUMO chains in SUMO^K14,30R, SUMO^D81R, or pli1Δ cells suppresses all tested STUbL mutant phenotypes (10, 28). Explaining this epistatic relationship, as demonstrated for Pli1, STUbL is only engaged when SUMO chains are present. Overall, these data provide direct mechanistic insight into the role of SUMO chains in STUbL targeting.

Ulp1 Protects Pli1 from Sumoylation and STUbL-mediated Degradation

Given that the Ulp1 SUMO protease is deregulated in nup132Δ cells, we tested whether increased Ulp1 dosage would impact the SUMO conjugation state and stability of Pli1. Strikingly, overexpression of Ulp1 in nup132Δ cells restored normal Pli1 levels (Fig. 4C). Moreover, Pli1 was restored in a hyposumoylated form, consistent with Ulp1 desumoylating and protecting it against STUbL-dependent degradation (Fig. 4C). Because budding yeast Ulp1 modulates transcription of the GAL1 gene (50), we monitored transcript levels of Pli1 upon Ulp1 overexpression. Whereas Ulp1 overexpression yields the anticipated increase in Ulp1 transcript levels, no difference in Pli1 transcription was detected (Fig. 4D). These results suggest that Ulp1 is normally optimally positioned to desumoylate and stabilize Pli1 and that increased Ulp1 dosage can compensate for this loss of spatial regulation.

Pli1 Overexpression Reverses SUMO-related Phenotypes of nup132Δ Cells

Pli1 acts at the end of the SUMO conjugation pathway to attach activated SUMO to its substrates. Therefore, if mature SUMO or E1/E2 factors were limiting, this would render the SUMO pathway unresponsive to the presence of Pli1. Therefore, to test whether Pli1 is the major or sole SUMO pathway factor deregulated by deleting Nup132, we overexpressed Pli1 in pli1Δ, nup132Δ, and nup132Δ slx8-29 cells. As anticipated, expression of Pli1 in pli1Δ control cells restored global SUMO conjugates to a level higher than that seen in wild type (Fig. 5A). Increased Pli1 dosage also restored SUMO conjugates in nup132Δ cells but to levels that were lower than those in pli1Δ cells (Fig. 5A). This is expected based on the constitutive degradation of Pli1 in nup132Δ but not pli1Δ cells. In slx8-29 nup132Δ double mutant cells, SUMO conjugates were higher than those in the nup132Δ single mutant upon Pli1 overexpression (Fig. 5A). Again, this is consistent with the role of STUbL in degrading Pli1 and antagonizing HMW SUMO conjugates. Overall, these data indicate that the SUMO pathway is largely intact upstream of Pli1 in nup132Δ cells.

FIGURE 5.

Pli1-related phenotypes in nup132Δ reverted by Pli1 expression. A, Western blots of SUMO conjugates or tubulin in the indicated strains in which Pli1 expression was induced (+), or not (−), from the nmt41 promoter for 24 h. The black arrowhead indicates the position of free mature SUMO. B, serial dilutions of the indicated strains transformed with empty control vector or pREP41-Pli1 that expresses Pli1 were spotted onto media without or with CPT grown at 35 °C. C and D, serial dilutions of the indicated strains were spotted onto rich (YES) or minimum selection (LAH) media at 32 °C, without or with the indicated drugs. YES, yeast extract, dextrose, amino acid supplement mixture; HU, hydroxyurea; CPT, camptothecin.

Thus far, our data indicate that the limited Pli1 activity present in nup132Δ cells rescues the lethality of slx8-29 cells. To directly test this, we assayed the effect of restoring Pli1 expression on the growth of slx8-29 nup132Δ cells. The growth of wild type or slx8-29 cells was similar whether they carried an empty vector control or Pli1 plasmid (Fig. 5B). As expected for the vector control (i.e. nup132Δ suppresses slx8-29), slx8-29 nup132Δ double mutant cells grew well compared with the slx8-29 single mutant (Fig. 5B). Notably, however, expression of Pli1 in slx8-29 nup132Δ cells caused poor growth and drug sensitivity that was similar to that of slx8-29 cells (Fig. 5B). Therefore, consistent with our biochemical analyses, restoring Pli1 expression is sufficient to nullify the rescue of slx8-29 phenotypes by nup132Δ.

We also tested whether, as was the case for cells lacking STUbL activity (28), deletion of Pli1 could suppress the growth and genome instability phenotypes of ulp2Δ cells. Indeed, the pli1Δ ulp2Δ double mutant phenocopies pli1Δ cells, indicating that pli1Δ is epistatic to ulp2Δ (Fig. 5C). We previously demonstrated that Pli1-dependent SUMO chain formation is toxic to both slx8-29 and ulp2Δ cells (10). Therefore, in keeping with our biochemical analysis, Pli1 degradation and reduction of HMW SUMO conjugates can explain the suppression of both slx8-29 and ulp2Δ phenotypes.

Compromised Centromere Function in Cells Lacking Nup132

Centromere function is disrupted in pli1Δ cells, whereby a ura4+ marker inserted at the heterochromatic inner repeats (imr) is constitutively expressed, versus the variegating phenotype in wild type cells (40). We therefore assessed chromatin function at imr in nup132Δ cells. As expected, wild type cells carrying the ura4+ insertion at imr (imr:ura4+) exhibited variegated expression, being able to grow both in the absence of uracil (leucine adenine histidine) and in the presence of 5-fluoroorotic acid (51). However, both pli1Δ imr:ura4+ and nup132Δ imr:ura4+ cells grew poorly in the presence of FOA but robustly in the absence of uracil (Fig. 5D). This result is consistent with the constitutive degradation of Pli1 in nup132Δ cells, causing hyposumoylation of key epigenetic regulators.

Discussion

Herein, we reveal a key novel consequence of nuclear pore dysfunction and subsequent spatial deregulation of Ulp1, which has a major impact on global sumoylation and centromere function. In budding yeast that lack Nup133 (SpNup132), Nup60, or Nup120, Ulp1 is destabilized and mislocalized in the nucleoplasm, causing altered global SUMO conjugate patterns and associated defects in genetic stability (16, 31, 35, 36, 52). A gain of Ulp1 function that promotes the desumoylation of normally spatially protected nucleoplasmic SUMO conjugates is proposed to cause these phenotypes. Such promiscuous desumoylation of nucleoplasmic SUMO conjugates by delocalized Ulp1 in nup132Δ fission yeast is also likely.

Interestingly, however, our results demonstrate that the residual Ulp1 activity in nup132Δ cells is unable to counteract Pli1 sumoylation. This leads to Pli1 degradation and associated hyposumoylation phenotypes. It will be interesting to determine whether this novel mechanism contributes to the SUMO conjugation defects reported for certain budding yeast nucleoporin mutants (16, 31, 35, 36, 52). In this regard, STUbL degrades the nuclear pool of a budding yeast PIAS family ligase, Siz1, particularly when its nuclear export is inhibited (53). Therefore, although untested, it is possible that nuclear pore-associated Ulp1 desumoylates Siz1 and protects it from degradation. If so, given the evolutionary divergence of fission and budding yeast, conservation of this mechanism in human cells seems likely.

How Ulp1, which is normally tethered at the nuclear periphery, accesses nucleoplasmic Pli1 is an interesting question for the future. We determined that increasing Ulp1 dosage in nup132Δ cells stabilized Pli1. This is consistent with increased Ulp1 activity compensating for the loss of a normally more coordinated interaction with Pli1. For example, fission yeast centromeres cluster at the nuclear periphery and are subject to functionally critical Pli1-dependent sumoylation (40, 54). Therefore, Pli1 acting at centromeres is also spatially organized at the nuclear periphery, more proximal to Ulp1.

In addition, budding yeast Ulp1 was recently shown to desumoylate transcription factors on chromatin to facilitate transcriptional activation, providing an example of recruitment of Ulp1 targets to the nuclear pore (50). Interestingly, a subcomplex of the human nuclear pore including Nup133 transiently localizes to centromeres and contributes to their mitotic function (55). Therefore, it will be interesting to determine whether Nup133-associated SENP2 (56) also modulates centromere/kinetochore sumoylation to support its function. Relocalization of the human pore complex and SUMO protease, rather than the target, may be a result of the open mitosis in human cells versus the closed mitosis of yeast.

Based on high throughput analysis, nup132Δ cells share another phenotype of pli1Δ cells, that is, highly elongated telomeres (40, 43). Notably, like centromeres, telomeres are also clustered at the nuclear periphery and are subject to Pli1-dependent regulation (40, 41). Pli1 sumoylates the telomere protein Tpz1 to support a mechanism for inhibiting excessive telomerase activity at chromosome ends (57). Therefore, based on the synonymous centromere silencing roles of Pli1 and Nup132, we envisage that Tpz1 will be hyposumoylated in cells lacking Nup132, leading to telomere elongation.

Identification of Pli1 as a STUbL substrate has enabled a comprehensive analysis of SUMO pathway and ancillary factor requirements. The role of SUMO chains in STUbL targeting has been largely inferred from (i) the accumulation of SUMO chain species when STUbL activity is compromised, (ii) the multivalent SUMO-interacting motif arrangement in STUbLs, and (iii) SUMO chain-induced autoubiquitination/degradation of RNF4 (25, 47, 58). We now demonstrate directly that the STUbL pathway is dependent on the chain forming lysine residues of SUMO, as well as the noncovalent SUMO-Ubc9 interface that promotes SUMO chain formation. These data fit well with the epistatic relationship of SUMO chain and STUbL mutants in fission yeast (10).

In addition, Cdc48-Ufd1-Npl4 was recently identified as a cofactor for STUbL in managing global SUMO conjugate levels (44, 45). However, evidence for a cooperative role in degrading a specific substrate was lacking. Herein, we show that both STUbL and Ufd1 are indeed involved in the proteasome-dependent turnover of Pli1 in nup132Δ cells.

As in fission yeast (44, 45), budding yeast Cdc48 also acts as a SUMO-targeted segregase that removes sumoylated DNA repair factors from chromatin (59). Given this degree of functional conservation, it seems likely that the human STUbL RNF4 and p97 cooperate in the remodeling of chromatin e.g. at DNA repair sites, as reported independently for each factor (60–63).

Author Contributions

M. N. and M. N. B. were both involved in study design, experimental execution, and writing of the manuscript.