The 2{micro}m Plasmid Causes Cell Death in Saccharomyces cerevisiae with a Mutation in Ulp1 Protease

The 2µm circle plasmid confers no phenotype in wild-typeSaccharomyces cerevisiae but in a nib1 mutant, an elevated plasmidcopy number is associated with cell death. Complementation wasused to identify nib1 as a mutant allele of the ULP1 gene thatencodes a protease required for removal of a ubiquitin-likeprotein, Smt3/SUMO, from protein substrates. The nib1 mutationreplaces conserved tryptophan 490 with leucine in the proteasedomain of Ulp1. Complete deletion of ULP1 is lethal, even ina strain that lacks the 2µm circle. Partial deletion ofULP1, like the nib1 mutation, results in clonal variations inplasmid copy number. In addition, a subset of these mutant cellsproduces lineages in which all cells have reduced proliferativecapacity, and this phenotype is dependent upon the presenceof the 2µm circle. Segregation of the 2µm circlerequires two plasmid-encoded proteins, Rep1 and Rep2, whichwere found to colocalize with Ulp1 protein in the nucleus andinteract with Smt3 in a two-hybrid assay. These associationsand the observation of missegregation of a fluorescently tagged2µm circle reporter plasmid in a subset of ulp1 mutantcells suggest that Smt3 modification plays a role in both plasmidcopy number control and segregation.

INTRODUCTION

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Introduction

The Saccharomyces cerevisiae 2µm circle is a rare exampleof a eukaryotic plasmid and has been exploited by molecularbiologists as the basis for most high-copy-number yeast vectors(14). Replication of the 2µm circle is coordinately regulatedwith replication of the host chromosomal DNA, and utilizes thehost cell enzymatic machinery, whereas segregation of the plasmidrequires the concerted action of the 2µm-encoded Rep1and Rep2 proteins and a cis-acting stability locus, STB, thatbears no resemblance to well-characterized yeast chromosomecentromeric sequences (6, 14). The 2µm circle also encodesa site-specific recombinase, Flp, that recognizes inverted repeattarget sites (FRT) within the plasmid and can increase copynumber if it falls below normal levels (approximately 60 copiesper haploid cell) (49). Futcher (15) has proposed that Flp-mediatedrecombination during DNA replication inverts one of the replicationforks, thereby allowing multimeric forms of the plasmid to beformed by a rolling circle mechanism from a single firing ofthe replication origin. Subsequent action of the Flp recombinasewould allow replication to terminate and resolve the multimersinto multiple monomeric copies of the plasmid. In support ofthis model, overexpression of the FLP gene leads to uncontrolledplasmid replication, which in turn causes longer cell cyclesand cell death, while combined overexpression of the Rep1 andRep2 proteins can repress the FLP gene and may limit amplificationof the plasmid once it has reached its normal copy number (35,39).

A mutation in the chromosomal NIB1 gene, nib1, leads to elevatedplasmid copy numbers, which suggests that plasmid levels mayalso be regulated by the host (17). We report here that NIB1is the ULP1 gene. The Ulp1 protein is a protease specific forremoval of a ubiquitin-like protein, Smt3/SUMO, from other proteinsto which it is covalently attached as a posttranslational modification(21, 23, 27, 28, 43). Ulp1 also converts the Smt3/SUMO primarytranslation product to the mature form that is conjugated totarget proteins (23). A second yeast Smt3-specific deconjugatingenzyme, Ulp2/Smt4, does not perform this essential processingfunction (24).

Smt3/SUMO modification apparently serves different functionsdepending on the substrate protein, either protecting the proteinfrom ubiquitin-mediated proteolysis as in the case of I ${kappa}$ B ${alpha}$ (8)or targeting the protein to a particular subcellular location.For example, SUMO modification of Ran GTPase-activating protein(RanGAP1) targets the otherwise cytosolic protein to the nuclearpore complex during interphase and to the spindle apparatusduring mitosis (28). Modification of PML (acute promyelocyticleukemia) protein is required for its localization to discretematrix-associated subnuclear domains, termed PML nuclear bodies(5, 11, 34).

Numerous SUMO-conjugated proteins have recently been identifiedand are involved in diverse cellular processes that includetranscription, chromatin remodeling, DNA replication and repair,metabolic processes, chromosome segregation, and cellular responsesto DNA damage and stress (19, 36, 50, 53). Different Smt3/SUMO-modifiedconjugates are observed at different cell cycle stages, suggestingthat cycling between conjugated and unmodified forms of targetproteins is cell cycle regulated (20, 23). Disruption of theUBC9 gene encoding the Smt3/SUMO-specific conjugating enzyme,Ubc9, leads to a G₂/M-phase cell cycle arrest (45), as doesdeletion of the ULP1 gene (23), indicating that modulating Smt3/SUMOmodification of target proteins is essential for normal cellcycle progression. We report here that in yeast with a partialdeletion of the ULP1 gene, as in nib1 mutants, 2µm plasmidlevels are elevated, implicating Ulp1-mediated Smt3 deconjugationin plasmid copy number control. Furthermore, a subset of cellsproduces lineages in which all cells cease proliferation. Thecolocalization of Rep1 and Rep2 with Ulp1 in the nucleus, theinteraction of both plasmid proteins with Smt3 in a two-hybridgenetic assay, and missegregation of a fluorescently tagged2µm circle reporter plasmid in a subset of ulp1 mutantcells suggest that Smt3 modification is involved in plasmidsegregation.

MATERIALS AND METHODS

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Materials and Methods

Strains and media. Yeast strains used in this study are given in Table 1. Strainslacking the 2µm circle, designated [cir⁰], were derivedfrom strains containing the 2µm circle, [cir⁺], eitherby first driving out the native 2µm circle with a selectable2µm-based plasmid and then screening for cells that hadlost the hybrid plasmid (9) or by expression of a defectiveFlp recombinase from plasmid pBIS-GALkFLP-(TRP1) (47). Standardmethods were used for growth and manipulation of yeast and bacteria(40, 42). Yeasts were grown in synthetic defined (SD) medium(0.67% Difco yeast nitrogen base without amino acids, 2% glucose,0.003% adenine, 0.002% uracil, and all required amino acids),or in YPD medium (1% yeast extract, 2% Bacto Peptone, 2% glucose)(40).

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TABLE 1. Yeast strains used in this study

DNA manipulations. A 5,130-bp BamHI restriction fragment containing the YPL020C/ULP1gene and YPL019C (an uncharacterized open reading frame [ORF]flanking ULP1) was amplified by PCR from CH568 genomic DNA withthe primers LPB1 (5'-CGGGATCCATATCCAATGATCTG-3') and LPB2 (5'-CGCGGATCCAACTTTCGAGTGGG-3')(details available on request) and cloned into the BamHI sitein the CEN/ARS/LEU2 yeast/Escherichia coli shuttle vector pRS315(46). Subsequently, the 2,872-bp BamHI/PstI fragment was subclonedinto BamHI/PstI doubly digested pRS315 to give pRS-ULP1. Complementationwas determined by transforming nib1 [cir⁺] strain CH569 to leucineprototrophy at the permissive temperature of 37°C and thenscoring colony morphology of transformants after growth at 30°C.Deletions of the chromosomal ULP1 gene were carried out by transformingyeast with an HpaI/BamHI restriction fragment from pRS-ULP1in which either the EcoRV/SalI fragment encoding the first 479bp (ulp1 ${Delta}$ ES) or an EcoRV/AvaII encoding the first 1,605 bp (ulp1 ${Delta}$ )of the 1,866-bp ULP1 ORF were replaced by standard DNA manipulationtechniques with a SmaI/SalI fragment encoding the yeast URA3gene. The URA3 gene was replaced by the LEU2 gene with a linearizedmarker swap plasmid as described by Cross (7). Gene deletionswere verified by PCR using the above primers and by using aPCR amplicon of the ULP1 ORF generated with primers LPB1 andLPB3 (LPB3, 5'-CGGAATTCATAATAATGTCAGTTG-3') as a radiolabeledprobe in a Southern blot analysis of genomic DNA isolated fromparent and transformed strains (42).

For two-hybrid analyses, the YPL020C ORF was PCR amplified usingprimers LPB1 and LPB3, digested with EcoRI and BamHI, and clonedin EcoRI/BamHI-digested pGAD424 (Clontech) to give pGAD-ULP1.The SMT3 ORF was amplified using primers 5'-CAGGATCCCGATGTCGGACTCA-3'and 5'-AACTGCAGCTAACCACCAATCTGTTCTC-3', digested with BamHIand PstI, and cloned in BamHI/PstI-digested pGAD424 to givepGAD-SMT3. In yeast, this plasmid directs expression of an in-framefusion of the Gal4 transcription activation domain (Gal4_AD)with the mature form of Smt3 lacking the three carboxy-terminalamino acids of the precursor Smt3 primary translation product(23). Two-hybrid plasmids directing expression of LexA-Rep fusionproteins have been previously described (44).

Plasmid copy number. For Southern blot determination of plasmid copy number, totalDNA was extracted from yeast growing logarithmically in SD mediumas previously described (10), digested with EcoRI, resolvedby electrophoresis on 1% agarose Tris-borate-EDTA gels, andtransferred to nylon membranes. A genomic 1.45-kb EcoRI fragmentencoding the TRP1 gene and a DNA fragment encoding the REP1ORF were ³²P labeled by random priming (41) and hybridized withthe membranes. Signals were quantified using a Bio-Rad phosphorimager,and the copy number of the 2µm plasmid was determinedby normalizing the 2µm signal to the TRP1 signal obtainedfrom the same DNA sample. For determination of plasmid copynumber relative to chromosomal content in colonies, a 2µmplasmid sequence and a chromosomal locus were amplified by PCRusing primers 5'-CCTTAACGGACCTACAG-3' and 5'-CGGGATCCTCGCATCCCCGGTT-3'and primers 5'-CTCTGGATCCGAATGAGTAAGCGGGGTAG-3' and 5'-CACCGGATCCGTAAAAGAACTTCTCCTC-3',respectively, from serial dilutions of total DNA isolated byglass bead breakage from suspensions of cells from coloniesof different sizes and morphologies (40). Amplicon yields weredetermined by densitometry of ethidium bromide-stained agarosegels on which the products had been resolved with MolecularAnalyst software from Bio-Rad.

Nuclear staining. Yeast cells growing logarithmically in YPD medium were harvested,washed in sterile phosphate-buffered saline (PBS) (137 mM NaCl,2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄), and fixed in 70%ethanol for 30 min at room temperature. Cells were then washedtwice in PBS, stained with 0.001% DAPI (4',6'-diamidino-2-phenylindole)(Sigma) in PBS for 15 min, washed again in PBS, and lightlysonicated to resolve clumping before being mounted on glassslides and photographed with a Leitz Laborlux visible UV-fluorescencemicroscope. All cells in randomly selected fields were scoredfor morphology and nuclear staining as described by Holm (17).For fluorescence-activated cell sorting, yeasts were fixed andstained with propidium iodide as described by Rose et al. (40).Cell cycle distribution was analyzed using Modfit LT for MacV3.0 software. Images were processed with Photoshop 5.5.

Immunofluorescence. The Ulp1 protein was Myc13 tagged at the carboxy terminus byhomologous integration at the chromosomal ULP1 locus of a Myc13-TRP1cassette (26). Yeast grown to mid-log phase were fixed, spheroplasted,and prepared for immunofluorescence staining essentially aspreviously described (38). Blocking was done using 1 mg ml^–1bovine serum albumin for 15 min. All primary and secondary antibodieswere diluted in PBS containing 1 mg ml^–1 bovine serumalbumin and 0.02% sodium azide. Incubations with primary andsecondary antibodies were done at room temperature for 60 and30 min, respectively, and visualized with a Leica confocal system,TCS4D. Rep1 and Rep2 polyclonal antisera have been describedpreviously (44).

Rep protein and plasmid localization. Green fluorescent protein (GFP)-Rep fusion proteins were expressedunder the control of the GAL10 promoter from the CEN/ARS plasmidspTS408-Rep1 and pTS408-REP2, respectively, as previously described(1). The TRP1-marked 2µm reporter plasmid, pSV5, containingthe 2µm circle origin of replication, STB locus, and 256repeats of the lac operator sequence (30) was visualized byexpressing GFP-LacI repressor fusion protein in yeast as previouslydescribed (48). Cells were observed with a Zeiss Axiovert 200fluorescence microscope (see Fig. 7A to C) and a Nikon invertedmicroscope with recommended excitation and emission filtersfor DAPI and GFP (see Fig. 7D and E). Images were captured withan Axiocam HRc camera and Axiovision 3.1 software or with aPhotometrics Quantix camera and Universal imaging Corp. Metamorphsoftware and analyzed using Adobe Photoshop 5.5.

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FIG. 7. Localization of Rep proteins and fluorescently tagged 2µm reporter plasmid in [cir⁺] ULP1 and ulp1 ${Delta}$ ES yeast strains. GFP-Rep1 (A), GFP-Rep2 (B), or GFP-LacI (C) fusion proteins were expressed in [cir⁺] ULP1 and ulp1 ${Delta}$ ES yeast strains. The yeasts shown in panel C were also transformed with the 2µm-derived reporter plasmid pSV5, containing lac operator repeats. Merged bright-field-GFP (left) and merged bright-field-DAPI (right) fluorescence images of representative cells are shown. [cir⁺] ULP1 (D) and [cir⁺] ulp1 ${Delta}$ ES (E) yeast cells expressing GFP-LacI and transformed with pSV5 were analyzed by time-lapse fluorescence microscopy. Bright-field and GFP fluorescence images of a single cell for each strain were taken every 6 min with the small-budded stage taken as the starting time point. Merged bright-field-GFP images are shown for all time points during the period when plasmids moved from mother to daughter cells and from representative earlier and subsequent times.

Two-hybrid assays. Transformants and cotransformants in the two-hybrid yeast hostCTY10/5d (3) were assayed for ß-galactosidase expressionby a permeabilized cell assay (40). Specificity of interactionswas confirmed by coexpressing LexA-Rep fusion proteins withGal4_AD-Snf4 fusion proteins (12) and Gal4_AD-Smt3 fusion proteinswith unfused LexA.

RESULTS

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Results

NIB1 is the ULP1 gene. Studies of yeast carrying the recessive nib1 mutation suggestthat host-encoded proteins may play a critical role in regulatingthe 2µm circle copy number. The nib1 mutation confersa "nibbled" colony morphology only in [cir⁺] yeast strains (Fig.1A), while nib1 [cir⁰] strains and wild type NIB1 [cir⁺] yeastform smooth colonies (17). The nibbled morphology arises dueto the production of lineages within the colony in which the2µm circle copy number is more than double normal levels(17). All further cells in that lineage eventually become mortal,arresting with a single large bud, a phenotype associated withyeast cell cycle mutants blocked in some aspect of DNA replicationor mitosis (16). The dead lineages are observed as "lethal sectors"in the growing colony. This nibbled phenotype was used to mapthe NIB1 gene to the left arm of chromosome XVI, tightly linkedto the RAD1 gene (18). To isolate the NIB1 gene, stretches ofDNA that contained ORFs in the vicinity of RAD1 were identifiedfrom the complete yeast genome sequence (Saccharomyces GenomeDatabase; http://www.yeastgenome.org/). Selected ORFs with theirflanking sequences were amplified by PCR using primers basedon the published sequence and cloned in a single-copy yeastLEU2/ARS/CEN vector, pRS315 (46). The resulting clones, containingthe YPL026C/STS1, YPL022W/RAD1, YPL018W/CTF19, YPL019C and YPL020C/ULP1genes, respectively, were tested for their ability to complementthe lethal sectoring phenotype when transformed into a nib1[cir⁺] yeast strain, CH569 (18). Only plasmids containing theULP1 gene were able to restore normal smooth-colony morphologyto the nib1 strain (Fig. 1A).

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FIG. 1. Colony morphology of wild-type and nib1 mutant yeasts. (A) Complementation of the nib1 "nibbled" colony morphology by the ULP1 gene. The nib1 [cir⁺] yeast strain CH569 was transformed with either the vector pRS315 or the plasmid pRS-ULP1, and the transformants were grown on solid SD medium lacking leucine at 30°C for 5 days. (B) Congenic haploid [cir⁺] and [cir⁰] yeast strains with either the wild-type ULP1 or mutant ulp1 ${Delta}$ ES allele were grown on solid YPD medium at 30°C for 5 days.

Mutations in ULP1 confer a 2µm-dependent nibbled colony phenotype. Deletion of the entire ULP1 ORF in either a [cir⁺] or [cir⁰]haploid yeast strain results in nonviability (23 and data notshown). Viable transformants were obtained in both [cir⁺] and[cir⁰] haploid yeast strains when only the first 479 bp of the1,866-bp genomic copy of the ULP1 ORF, including the initiatingmethionine codon, were replaced with the URA3 gene running inan antisense orientation (Fig. 1B). Although the [cir⁺] transformantswith this partial deletion of the ULP1 gene (hereafter referredto as the ulp1 ${Delta}$ ES allele) were viable, the colonies had a nibbledmorphology, similar to that of the CH569 [cir⁺] nib mutants(Fig. 1A) (18), whereas the [cir⁰] ulp1 ${Delta}$ ES yeast had normal colonymorphology (Fig. 1B). This dependence of the lethal sectoringphenotype on the presence of the 2µm plasmid and the abilityof the wild-type YPL020C ORF to complement the nib1 defect supportthe identification of ULP1 as the NIB1 gene. Sequencing of theULP1 ORF from the original CH569 [cir⁺] nib1 mutant yeast strainrevealed a single base change from the wild-type sequence, aG-to-T transversion at position 1469. This mutation would resultin a nonconservative substitution of a leucine for a tryptophanresidue at position 490 in the Ulp1 protein, a mutation previouslyreported as conferring a temperature-sensitive phenotype inyeast (33).

Both [cir⁰] and [cir⁺] ulp1 ${Delta}$ ES yeast mutants lag at G₂/M phase of the cell cycle. To further characterize the defect conferred by the ulp1 ${Delta}$ ES allele,the nuclear and cellular morphology of [cir⁺] and [cir⁰] yeastcarrying either the mutant or wild-type ULP1 gene was examined(Table 2). In a logarithmically growing culture, 28% of [cir⁺]ulp1 ${Delta}$ ES mutant cells had a large bud and a single nucleus atthe bud neck, compared to 7% for the congenic [cir⁺] ULP1 strain.These results are similar to those previously reported for [cir⁺]nib1 mutant yeast (17) and support the involvement of the encodedprotein in cell cycle progression (23). Surprisingly, the [cir⁰]ulp1 ${Delta}$ ES yeast mutant also showed a distribution of cellular morphologiessimilar to that obtained with the [cir⁺] ulp1 ${Delta}$ ES mutant, despitethe apparent lack of effect of this mutation on colony morphology(Fig. 1B). The results suggest that both [cir⁺] and [cir⁰] ulp1 ${Delta}$ EScells are delayed either in DNA replication or in the G₂/M stagein the cell cycle. The presence of the 2µm circle seemsto exacerbate the defect, with a higher percentage of anucleatecells being observed with the [cir⁺] ulp1 ${Delta}$ ES culture than withthe others. These anucleate cells suggest cytokinesis may haveoccurred despite mitosis not having been properly executed.The [cir⁺] ulp1 ${Delta}$ ES cells were often enlarged, had elongated buds,and displayed an apical chain-like budding pattern, phenotypessimilar to those produced by overexpression of FLP in a [cir⁺]yeast strain (35).

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TABLE 2. Cell morphology and nuclear staining of ULP1 and ulp1 ${Delta}$ ES yeast^a

To establish whether the cell cycle lag observed with the ulp1 ${Delta}$ ESyeast was due to a delay in DNA replication or was at the G₂/Mboundary, the cells were analyzed by flow cytometry (Fig. 2).The [cir⁺] and [cir⁰] ULP1 cells both displayed the distributionexpected for a population of unsynchronized wild-type cells,with approximately half the cells having the 2N DNA contentof G₂, 40% having the 1N DNA content of G₁, and the remainderundergoing DNA replication (S). In contrast, a higher percentageof the population for both [cir⁰] and [cir⁺] ulp1 ${Delta}$ ES yeast mutantshad a 2N DNA content, consistent with the cell cycle delay atthe G₂/M boundary. This was most pronounced for the [cir⁺] ulp1 ${Delta}$ ESyeast strain where almost no cells with a 1N DNA content wereobserved. In addition, a significant proportion of the [cir⁺]ulp1 ${Delta}$ ES yeast cells had a DNA content higher than expected fora 2N chromosome complement.

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FIG. 2. G₂/M lag is exacerbated in [cir⁺] ulp1 ${Delta}$ ES yeast. Unsynchronized cultures of congenic ULP1 and ulp1 ${Delta}$ ES yeast, [cir⁺] and [cir⁰], were grown to early log phase in YPD medium, and the cells were fixed, stained with propidium iodide, visualized by fluorescence microscopy (left), and analyzed by flow cytometry (center), with the percentage of cells at each phase of the cell cycle and the percentage of aberrant cells (Ab), those with DNA content greater than 2N or less than 1N, shown at right. Scale bar, 5 µm.

Yeasts with partial deletion of ULP1 have elevated 2µm plasmid levels. Consistent with the greater than diploid DNA content observedby flow cytometry, [cir⁺] yeast cells carrying the ulp1 ${Delta}$ ES allelewere found by Southern blotting to have a two- to fivefold amplificationof the 2µm plasmid copy number relative to their parentalULP1 strain (Fig. 3 and data not shown). This amplificationis similar to that previously observed with [cir⁺] nib1 mutants(17). Copy numbers for independent cultures of a given transformantvaried. In addition, smooth colonies occasionally arose spontaneouslyin the [cir⁺] ulp1 ${Delta}$ ES strain and were found by Southern blottingto have become [cir⁰] (data not shown). This clonal variabilityin plasmid copy number suggests a stochastic process in whichsome cells acquire either higher- or lower-than-average numbersof plasmid molecules at cellular division and that the frequencyof these events may be increased in ulp1 ${Delta}$ ES yeast.

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FIG. 3. Amplification of 2µm circle copy number in ulp1 ${Delta}$ ES yeast. Southern blot analysis of EcoRI-restricted genomic DNA isolated from the indicated yeast strains. Phosphorimages of the blots after sequential hybridization with radiolabeled probes recognizing either the genomic TRP1 locus or the 2µm circle (the 2µm circle exists in equimolar amounts of two forms, giving the bands detected with this probe) are shown. TRP1-probed images are longer exposures to allow the weaker signal from this single-copy genomic probe to be visualized.

Commitment to cease proliferation is inherited. What remains unexplained is why the presence of the 2µmcircle in ulp1 ${Delta}$ ES yeast results in clonal lethality of a subpopulationof the cells, observed as a nibbled colony morphology. Lethalsectors would not be produced if random cells in the colonyarrested as a consequence of the mutation. Proliferation ofneighboring cells would obscure their loss. Instead, cells thatproduce lethal sectors must continue to proliferate after theyhave become committed to death, pass their commitment on totheir progeny, and then die during a later cell division. Totest this hypothesis, individual [cir⁺] ulp1 ${Delta}$ ES and ULP1 cellswere assessed by a pedigree assay for their colony-forming ability(Table 3). As expected, all cells in the completed [cir⁺] ULP1pedigrees produced normal large smooth colonies. Similarly,for over half of all pedigrees completed for the [cir⁺] ulp1 ${Delta}$ ESmutant, all cells within the pedigree produced large but (inthis case) variably nibbled colonies. In contrast, for aboutone-quarter of the completed [cir⁺] ulp1 ${Delta}$ ES mutant pedigrees,all cells produced microcolonies of <100 cells. In the latterpedigrees, the results suggest that the initial mother cellhad already become committed to cease proliferation and thatdaughter and the granddaughter cells inherited the commitment.Commitment might reflect an elevated plasmid copy number inthese mother cells.

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TABLE 3. Pedigree analysis of colony-forming ability of ULP1 and ulp1 ${Delta}$ ES [cir⁺] yeast^a

The 2µm circle exacerbates the effect of the ulp1 ${Delta}$ ES mutation on growth. To further investigate the basis of the ulp1 ${Delta}$ ES defect, we comparedthe rate of growth of [cir⁰] and [cir⁺] ulp1 ${Delta}$ ES strains to ULP1yeast in liquid culture (Fig. 4). Both the [cir⁰] and [cir⁺]ulp1 ${Delta}$ ES strains underwent a diauxic shift and entered stationaryphase at a lower cell density than their respective ULP1 strains.Since the average size of a ulp1 ${Delta}$ ES cell is greater than thatof a ULP1 cell (Fig. 2), the lower cell density at diauxic shiftmay result from the larger mutant cells, exhausting the glucoseat a lower cell density, irrespective of their plasmid content.In addition to an earlier entry into stationary phase, the [cir⁺]ulp1 ${Delta}$ ES strain grew significantly slower during the logarithmicphase than either the [cir⁰] or [cir⁺] ULP1 strains or the [cir⁰]ulp1 ${Delta}$ ES mutant strain. This growth difference is consistent withthe [cir⁺] ulp1 ${Delta}$ ES strain having a colony morphology that differsfrom that of the others. The reduced rate of logarithmic growthin the [cir⁺] ulp1 ${Delta}$ ES yeast may represent the production of cellsthat constitute the lethal sectors in colonies formed by thesestrains.

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FIG. 4. The 2µm plasmid impairs growth of ulp1 ${Delta}$ ES yeast. Yeast cells were grown to stationary phase in liquid SD medium, diluted into fresh medium, and incubated with shaking at 30°C. Initial and subsequent cell densities at the indicated times after dilution were measured by hemocytometer counts. Results are the averages with standard deviations for four cultures of each strain.

Plasmid copy number varies with colony size and morphology. Replating of [cir⁺] ulp1 ${Delta}$ ES yeast cells obtained from a nibbledcolony results in a variety of different colony sizes rangingfrom large normal-sized colonies to microcolonies of <100cells and colony morphologies ranging from smooth to extensivelynibbled (Fig. 5A). Cell size and shape are also more variablefor the [cir⁺] ulp1 ${Delta}$ ES yeast, even for cells within a singlecolony (Fig. 5B). To determine whether the different-sized andshaped colonies observed with [cir⁺] ulp1 ${Delta}$ ES yeast reflecteddifferences in plasmid levels within the cells of the colony,a semiquantitative PCR approach was undertaken (Table 4). Forthe DNA isolated from nibbled ulp1 ${Delta}$ ES mutant colonies, 2µmplasmid levels showed a trend of increasing with decreasingcolony size relative to the level of 2µm plasmid in aULP1 strain. The smooth ulp1 ${Delta}$ ES mutant colonies had only a twofoldincrease in 2µm plasmid levels. This less-dramatic increasein plasmid copy number might explain the loss of the nibbledcolony phenotype. The correlation between the increase in 2µmplasmid levels and the loss of proliferative capacity, as reflectedin reduced colony size, supports the hypothesis that elevatedplasmid levels lead to cell death in ulp1 ${Delta}$ ES yeast. Consistentwith this hypothesis is the observation that [cir⁰] ulp1 ${Delta}$ ES yeastdoes not produce nibbled colonies or microcolonies.

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FIG. 5. Variable cell and colony size and morphology in [cir⁺] ulp1 ${Delta}$ ES yeast. (A) [cir⁰] and [cir⁺] ulp1 ${Delta}$ ES yeast mutants were grown on solid SD medium for 5 days. Colony size and morphology varied only for the [cir⁺] strain. Several large- and medium-sized nibbled colonies, one large smooth colony, and a small nibbled colony are shown. (B) Bright-field images (magnification, x100) are of a [cir⁰] ulp1 ${Delta}$ ES colony (left) and [cir⁺] ulp1 ${Delta}$ ES yeast colonies (right), with one small nibbled colony adjacent to a large nibbled colony, from the respective plates above in panel A.

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TABLE 4. Variation in plasmid levels in colonies with different morphologies

Ulp1 colocalizes in the nucleus with the 2µm circle-encoded Rep proteins. The possible role of the ULP1 gene in the interplay betweenthe physiology of the nuclear resident plasmid and the cellcycle of its host led us to compare the intracellular distributionof the Ulp1 protein and the 2µm circle-encoded Rep1 andRep2 proteins. Immunofluorescence staining of the Ulp1 proteintagged with the Myc epitope and expressed from the native ULP1promoter showed that nearly all of the protein was internalto but not uniformly distributed within the nucleus (Fig. 6).This is in agreement with previous data showing that Ulp1 concentratedin the nuclear periphery-nuclear envelope region (24). Quitestrikingly, there was near-perfect colocalization between theUlp1 protein and the 2µm circle-encoded Rep proteins,with the Rep2 distribution in particular being indistinguishablefrom that of Ulp1.

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FIG. 6. In situ localization of the Ulp1 and Rep proteins. The Ulp1 protein carrying a carboxy-Myc13 tag was expressed from the native promoter at the ULP1 chromosomal locus. A Myc-specific monoclonal antibody conjugated to rhodamine was used to localize the protein while DNA was stained with DAPI. Native Rep1 (A) and native Rep2 (B) proteins were detected with Rep protein-specific primary polyclonal antibodies and visualized with secondary antibodies conjugated to FITC.

In vivo interaction of 2µm-encoded Rep proteins with Smt3. The apparent colocalization of the 2µm circle-encodedRep proteins with the Ulp1 protease suggested there might bea direct interaction between these proteins in the cell. A two-hybridassay was used to test for this association (3). No interactionwas detected between the Rep proteins and Ulp1 (data not shown),but both Rep1 and Rep2 gave specific interactions with Smt3,the ubiquitin-like protein removed from target proteins by theUlp1 protease (Table 5) (23). The interaction suggests thatthe 2µm Rep proteins may either be modified by Smt3 orassociate with the Smt3-modified protein(s).

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TABLE 5. Two-hybrid interaction of Smt3 with Rep1 and Rep2

Rep protein localization in ulp1 ${Delta}$ ES yeast. The Rep proteins have been shown to colocalize with the 2µmcircle in the nucleus (48). To determine whether this localizationwas affected by the ulp1 ${Delta}$ ES mutation, GFP-tagged versions ofRep1 (Fig. 7A) and Rep2 (Fig. 7B) were expressed in yeast. BothRep fusion proteins colocalized with the DAPI-stained chromosomalDNA in the ulp1 ${Delta}$ ES mutant cells. The nuclear distribution ofthe plasmid in the mutant did not significantly differ fromthat observed with ULP1 cells, indicating that the increasein plasmid copy number in the ulp1 ${Delta}$ ES mutant was not due to alteredlocalization of Rep1 and Rep2.

Plasmid segregation in ulp1 ${Delta}$ ES yeast. To determine whether 2µm plasmid distribution was affectedby the ulp1 ${Delta}$ ES mutation, a 2µm-derived reporter plasmid,pSV5, containing multiple lac operator sequences was visualizedin cells expressing a GFP-LacI repressor fusion protein (48).The fluorescently tagged 2µm plasmids appeared as a smallnumber of discrete foci colocalized with the DAPI-stained chromosomalDNA in both [cir⁺] ulp1 ${Delta}$ ES and [cir⁺] ULP1 cells (Fig. 7C), indicatingthat plasmid localization within the nucleus did not requirewild-type ULP1 function. Although the clustering of the plasmidin foci within the nucleus was unaffected, GFP-LacI fluorescencewas not equally intense in some mother and daughter ulp1 ${Delta}$ ES mutantcells, suggesting that the 2µm-derived reporter plasmidwas not equally partitioned during mitosis (Fig. 7C). To assessthis difference, cells with large buds having two distinct DAPI-stainedmasses, consistent with the mother cell having completed mitosis,were scored for their relative intensity of GFP-LacI repressorfluorescence (Table 6). Complete missegregation of the 2µm-derivedreporter plasmid was observed with 6% of [cir⁺] ulp1 ${Delta}$ ES mutantcells and not observed with [cir⁺] ULP1 cells, suggesting thatthe mutation in ULP1 impaired 2µm circle plasmid segregation.

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TABLE 6. Distribution of fluorescently tagged 2µm reporter plasmid in ULP1 and ulp1 ${Delta}$ ES yeast

To further characterize the plasmid segregation defect, inheritanceof the fluorescently tagged 2µm reporter plasmid was monitoredin live cells by time-lapse photography (Fig. 7D and 7E). Plasmiddelivery to the daughter cell took <6 min in the [cir⁺] ULP1cells, with GFP-LacI repressor fluorescence equally partitionedbetween mother and daughter cells. In the [cir⁺] ulp1 ${Delta}$ ES cell,the fluorescently tagged plasmids took >12 min to move frommother to daughter cell, and in some cells, all fluorescencewas delivered to and retained in the daughter cell, consistentwith complete missegregation of the plasmid in these mitoticdivisions. The longer time taken for plasmid segregation inthe [cir⁺] ulp1 ${Delta}$ ES mutant is consistent with the G₂/M lag observedfor these mutant cells by flow cytometry (Fig. 2). The directionof plasmid missegregation contrasts with the maternal bias reportedfor plasmids that lack a functional partitioning system (6).However, more plasmid missegregation events need to be capturedby time-lapse microscopy to establish whether missegregationis random or biased toward the daughter cell in the ulp1 ${Delta}$ ES mutant.

DISCUSSION

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Discussion

The discovery that nib1, a mutation that results in elevatedplasmid copy numbers, is a single amino acid change in a proteaserequired for cell cycle progression provides another host functionthat the 2µm circle requires for its stable maintenancewithin the yeast nucleus. Partial deletion of the ULP1 geneencoding the protease was found to mimic the nib1 mutation (17).The G₂/M lag in both [cir⁺] and [cir⁰] yeast strains with partialdeletion of the ULP1 gene is consistent with the G₂/M arrestobserved for yeasts carrying a complete deletion of the ULP1ORF (23). In our study, the ulp1 ${Delta}$ ES allele retained the 3' endof the ORF encoding the conserved protease domain (23) (33)and part of the noncatalytic regulatory domain required forretention of Ulp1 at nuclear pores (37). The remarkable similaritybetween the phenotypes conferred by this deletion allele andby nib1, a mutation that alters a single amino acid within theconserved carboxy-terminal protease domain, suggests that theulp1 ${Delta}$ ES allele expresses a truncated protein with protease activitysufficient for viability, but insufficient for normal plasmidmaintenance. Both alleles may decrease the availability of freemature Smt3, either by impairing the proteolytic activity orby reducing expression of Ulp1. The loss of Ulp1 processingactivity would reduce the level of some Smt3 conjugates, whileother targets that are normally deconjugated by Ulp1 could accumulate(23, 25). The altered levels of these modified target proteinsmay be responsible for the phenotype of elevated 2µm circleplasmid levels common to both mutant ulp1 alleles.

Although not specifically indicated, yeast strains used in previousexperiments where either the ULP1 or UBC9 genes were disruptedor deleted (23, 45) would, like most strains of Saccharomycescerevisae, probably be [cir⁺]. Altered levels of the 2µmcircle may occur in these mutant yeasts. The distinct domainsof nuclear staining with propidium iodide, observed when yeastscarrying temperature-sensitive alleles of the ULP1 gene wereshifted to and incubated at the nonpermissive temperature (23),may correspond to elevated plasmid levels.

Deletion of the SMT4/ULP2 gene encoding the second yeast Smt3-deconjugatingprotease, Ulp2 (24), or simultaneous deletion of MLP1 and MLP2,two genes encoding myosin-like proteins required for nuclearpore localization and stabilization of Ulp1 (52), also resultsin abnormal cell morphology, temperature-sensitive growth, andformation of nibbled colonies in [cir⁺] yeast, phenotypes remarkablysimilar to the 2µm circle-associated phenotypes observedwith the ulp1 ${Delta}$ ES mutant yeast. Accumulation of the 2µmcircle in these mutant cells may also occur in response to lossor mislocalization of Smt3-deconjugating activity.

The lethality observed with [cir⁺] ulp1 ${Delta}$ ES mutant yeast correlateswith elevated plasmid copy numbers and may reflect titrationof a host protein critical for progression through mitosis.Increased plasmid levels in the ulp1 mutant could arise fromenhanced Flp recombinase-mediated amplification or from plasmidmissegregation. Although plasmid missegregation seems more likelyin light of the observed missegregation of the fluorescentlytagged 2µm reporter plasmid, it is possible that alteredFlp activity is the primary defect and that plasmid missegregationis a secondary response to elevated plasmid copy number. AlteredSmt3 modification could stabilize the Flp protein or reduceRep protein-mediated repression of FLP gene expression. In thealternative model, altered levels of a Smt3-modified protein(s)common to the pathways of both chromosome and plasmid segregationcould be responsible for the G₂/M cell cycle delay in the [cir⁰]ulp1 ${Delta}$ ES mutant yeast and the missegregation of the multicopyplasmid observed with some [cir⁺] ulp1 ${Delta}$ ES mutant cells. The higher-than-normalnumber of plasmids received by some cells could further impairplasmid segregation in subsequent divisions due to increasedcompetition for limiting Smt3-conjugates. In cells that receivedless plasmid, the Flp recombinase would restore the normal plasmidcopy number, the result being a continuous increase in plasmidlevels in the culture.

Smt3 modification has been implicated in regulating chromosomesegregation. The SMT3 gene was originally cloned on the basisof its ability, when overexpressed, to suppress the effectsof mutation in Mif2, the yeast homolog of mammalian CENP-C,a kinetochore protein required for chromosome segregation andmitotic spindle integrity (31, 32). CENP-C may itself be SUMOmodified (13). Smt3 modification may also participate in chromosomesegregation by contributing to cohesion between chromosomes.Mcd1 and Smc1, two components of cohesin, a complex enrichedat chromosomal centromeres that holds sister chromatids togetheruntil the onset of anaphase, are Smt3 modified (4, 22, 50).In a ulp2 mutant, centromeric cohesion is impaired in responseto altered Smt3 modification of topoisomerase II (2), and asimilar defect might be produced by loss of Ulp1 function. SinceSUMO modification targets RanGAP1 to the spindle apparatus duringmitosis (28), it is tempting to speculate that centromeric cohesincomponents and the 2µm circle use a similar approach,with Smt3/SUMO modification mediating attachment to the hostspindle and conferring efficient segregation. In support ofthis hypothesis, the mitotic spindle has recently been shownto promote recruitment of the cohesin complex to the 2µmcircle STB-partitioning locus (29).

The colocalization of the Ulp1 protease with the 2µm circle-encodedRep proteins and the two-hybrid interaction of both Rep1 andRep2 with Smt3 suggested the plasmid proteins might be targetsof Smt3 conjugation or interact with Smt3-modified proteins.Evidence for Rep2, Flp, and possibly Rep1 being Smt3 modifiedhas recently been obtained (E. Johnson, personal communication).The Rep proteins are essential for normal plasmid segregationat cytokinesis, are tightly associated with the 2µm circleDNA in a small number of discrete foci in live yeasts (48),and probably form a DNA-protein aggregate at the plasmid STBlocus (6, 44). Efficient plasmid segregation involves recruitmentof cohesin complexes to STB by the Rep proteins (30, 51). Cyclingbetween Smt3-modified and unmodified forms of the plasmid proteinscould allow their retention or release from a nuclear substructurein response to cell cycle signals, a process that may mediateboth efficient plasmid segregation and copy number control.In the ulp1 ${Delta}$ ES mutant, altered levels of Smt3-conjugated Repproteins or cohesin components may affect plasmid associationwith the host spindle or impair separation of the 2µmplasmid-containing foci, structures that may colocalize witha yeast equivalent of the mammalian matrix-associated PML nuclearbodies (5, 11, 34). Identifying the targets of Ulp1 proteaseat the G₂/M stage in the cell cycle may help reveal the mechanismby which a simple extrachromosomal DNA element parasitizes componentsof the sophisticated host cell cycle apparatus for its own stable,high-copy-number propagation.

ACKNOWLEDGMENTS

We thank C. Holm for the gift of yeast strains CH568 and CH569,Marc Gartenberg for the plasmid pBIS-GALkFLP-(TRP1), BarbaraGoettgens (Core Facility, Institute for Cell and Molecular Biology,University of Texas, Austin) for assistance with confocal microscopy,and J. Benjamin and Y. Zhang for technical assistance. We aregrateful to Erica Johnson for communicating results prior topublication and for helpful comments on the manuscript.

This work was supported by an NSERC grant to M.J.D. and grantsfrom the National Institutes of Health and the Robert WelchFoundation to M.J.

FOOTNOTES

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, Faculty of Medicine, Dalhousie University, Sir Charles Tupper Medical Building, Halifax, Nova Scotia, Canada B3H 1X5. Phone: (902) 494-7182. Fax: (902) 494-1355. E-mail: dobson{at}dal.ca.

REFERENCES

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