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Previous Article | Next Article 
Molecular and Cellular Biology, May 2005, p. 4299-4310, Vol. 25, No. 10
0270-7306/05/$08.00+0 doi:10.1128/MCB.25.10.4299-4310.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Department of Biochemistry and Molecular Biology, Faculty of Medicine, Dalhousie University, Sir Charles Tupper Medical Building, Halifax, Nova Scotia, Canada B3H 1X5,1 Department of Microbiology, University of Texas at Austin, Austin, Texas 787122
Received 7 October 2004/ Returned for modification 20 November 2004/ Accepted 13 February 2005
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ABSTRACT |
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INTRODUCTION |
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A mutation in the chromosomal NIB1 gene, nib1, leads to elevated plasmid copy numbers, which suggests that plasmid levels may also be regulated by the host (17). We report here that NIB1 is the ULP1 gene. The Ulp1 protein is a protease specific for removal of a ubiquitin-like protein, Smt3/SUMO, from other proteins to which it is covalently attached as a posttranslational modification (21, 23, 27, 28, 43). Ulp1 also converts the Smt3/SUMO primary translation product to the mature form that is conjugated to target proteins (23). A second yeast Smt3-specific deconjugating enzyme, Ulp2/Smt4, does not perform this essential processing function (24).
Smt3/SUMO modification apparently serves different functions depending on the substrate protein, either protecting the protein from ubiquitin-mediated proteolysis as in the case of I
B
(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 nuclear pore complex during interphase and to the spindle apparatus during mitosis (28). Modification of PML (acute promyelocytic leukemia) protein is required for its localization to discrete matrix-associated subnuclear domains, termed PML nuclear bodies (5, 11, 34).
Numerous SUMO-conjugated proteins have recently been identified and are involved in diverse cellular processes that include transcription, chromatin remodeling, DNA replication and repair, metabolic processes, chromosome segregation, and cellular responses to DNA damage and stress (19, 36, 50, 53). Different Smt3/SUMO-modified conjugates are observed at different cell cycle stages, suggesting that cycling between conjugated and unmodified forms of target proteins is cell cycle regulated (20, 23). Disruption of the UBC9 gene encoding the Smt3/SUMO-specific conjugating enzyme, Ubc9, leads to a G2/M-phase cell cycle arrest (45), as does deletion of the ULP1 gene (23), indicating that modulating Smt3/SUMO modification of target proteins is essential for normal cell cycle progression. We report here that in yeast with a partial deletion of the ULP1 gene, as in nib1 mutants, 2µm plasmid levels are elevated, implicating Ulp1-mediated Smt3 deconjugation in plasmid copy number control. Furthermore, a subset of cells produces lineages in which all cells cease proliferation. The colocalization of Rep1 and Rep2 with Ulp1 in the nucleus, the interaction of both plasmid proteins with Smt3 in a two-hybrid genetic assay, and missegregation of a fluorescently tagged 2µm circle reporter plasmid in a subset of ulp1 mutant cells suggest that Smt3 modification is involved in plasmid segregation.
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MATERIALS AND METHODS |
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ES) or an EcoRV/AvaII encoding the first 1,605 bp (ulp1) of the 1,866-bp ULP1 ORF were replaced by standard DNA manipulation techniques with a SmaI/SalI fragment encoding the yeast URA3 gene. The URA3 gene was replaced by the LEU2 gene with a linearized marker swap plasmid as described by Cross (7). Gene deletions were verified by PCR using the above primers and by using a PCR amplicon of the ULP1 ORF generated with primers LPB1 and LPB3 (LPB3, 5'-CGGAATTCATAATAATGTCAGTTG-3') as a radiolabeled probe in a Southern blot analysis of genomic DNA isolated from parent and transformed strains (42). For two-hybrid analyses, the YPL020C ORF was PCR amplified using primers LPB1 and LPB3, digested with EcoRI and BamHI, and cloned in 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 BamHI and PstI, and cloned in BamHI/PstI-digested pGAD424 to give pGAD-SMT3. In yeast, this plasmid directs expression of an in-frame fusion of the Gal4 transcription activation domain (Gal4AD) with the mature form of Smt3 lacking the three carboxy-terminal amino acids of the precursor Smt3 primary translation product (23). Two-hybrid plasmids directing expression of LexA-Rep fusion proteins have been previously described (44).
Plasmid copy number. For Southern blot determination of plasmid copy number, total DNA was extracted from yeast growing logarithmically in SD medium as previously described (10), digested with EcoRI, resolved by electrophoresis on 1% agarose Tris-borate-EDTA gels, and transferred to nylon membranes. A genomic 1.45-kb EcoRI fragment encoding the TRP1 gene and a DNA fragment encoding the REP1 ORF were 32P labeled by random priming (41) and hybridized with the membranes. Signals were quantified using a Bio-Rad phosphorimager, and the copy number of the 2µm plasmid was determined by normalizing the 2µm signal to the TRP1 signal obtained from the same DNA sample. For determination of plasmid copy number relative to chromosomal content in colonies, a 2µm plasmid sequence and a chromosomal locus were amplified by PCR using 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 by glass bead breakage from suspensions of cells from colonies of different sizes and morphologies (40). Amplicon yields were determined by densitometry of ethidium bromide-stained agarose gels on which the products had been resolved with Molecular Analyst 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 washed twice in PBS, stained with 0.001% DAPI (4',6'-diamidino-2-phenylindole) (Sigma) in PBS for 15 min, washed again in PBS, and lightly sonicated to resolve clumping before being mounted on glass slides and photographed with a Leitz Laborlux visible UV-fluorescence microscope. All cells in randomly selected fields were scored for morphology and nuclear staining as described by Holm (17). For fluorescence-activated cell sorting, yeasts were fixed and stained with propidium iodide as described by Rose et al. (40). Cell cycle distribution was analyzed using Modfit LT for Mac V3.0 software. Images were processed with Photoshop 5.5.
Immunofluorescence. The Ulp1 protein was Myc13 tagged at the carboxy terminus by homologous integration at the chromosomal ULP1 locus of a Myc13-TRP1 cassette (26). Yeast grown to mid-log phase were fixed, spheroplasted, and prepared for immunofluorescence staining essentially as previously described (38). Blocking was done using 1 mg ml–1 bovine serum albumin for 15 min. All primary and secondary antibodies were diluted in PBS containing 1 mg ml–1 bovine serum albumin and 0.02% sodium azide. Incubations with primary and secondary antibodies were done at room temperature for 60 and 30 min, respectively, and visualized with a Leica confocal system, TCS4D. Rep1 and Rep2 polyclonal antisera have been described previously (44).
Rep protein and plasmid localization. Green fluorescent protein (GFP)-Rep fusion proteins were expressed under the control of the GAL10 promoter from the CEN/ARS plasmids pTS408-Rep1 and pTS408-REP2, respectively, as previously described (1). The TRP1-marked 2µm reporter plasmid, pSV5, containing the 2µm circle origin of replication, STB locus, and 256 repeats of the lac operator sequence (30) was visualized by expressing GFP-LacI repressor fusion protein in yeast as previously described (48). Cells were observed with a Zeiss Axiovert 200 fluorescence microscope (see Fig. 7A to C) and a Nikon inverted microscope with recommended excitation and emission filters for DAPI and GFP (see Fig. 7D and E). Images were captured with an Axiocam HRc camera and Axiovision 3.1 software or with a Photometrics Quantix camera and Universal imaging Corp. Metamorph software and analyzed using Adobe Photoshop 5.5.
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RESULTS |
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ES allele) were viable, the colonies had a nibbled morphology, similar to that of the CH569 [cir+] nib mutants (Fig. 1A) (18), whereas the [cir0] ulp1ES yeast had normal colony morphology (Fig. 1B). This dependence of the lethal sectoring phenotype on the presence of the 2µm plasmid and the ability of the wild-type YPL020C ORF to complement the nib1 defect support the identification of ULP1 as the NIB1 gene. Sequencing of the ULP1 ORF from the original CH569 [cir+] nib1 mutant yeast strain revealed a single base change from the wild-type sequence, a G-to-T transversion at position 1469. This mutation would result in a nonconservative substitution of a leucine for a tryptophan residue at position 490 in the Ulp1 protein, a mutation previously reported as conferring a temperature-sensitive phenotype in yeast (33).
Both [cir0] and [cir+] ulp1ES yeast mutants lag at G2/M phase of the cell cycle.
To further characterize the defect conferred by the ulp1ES allele, the nuclear and cellular morphology of [cir+] and [cir0] yeast carrying either the mutant or wild-type ULP1 gene was examined (Table 2). In a logarithmically growing culture, 28% of [cir+] ulp1ES mutant cells had a large bud and a single nucleus at the 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 encoded protein in cell cycle progression (23). Surprisingly, the [cir0] ulp1ES yeast mutant also showed a distribution of cellular morphologies similar to that obtained with the [cir+] ulp1ES mutant, despite the apparent lack of effect of this mutation on colony morphology (Fig. 1B). The results suggest that both [cir+] and [cir0] ulp1ES cells are delayed either in DNA replication or in the G2/M stage in the cell cycle. The presence of the 2µm circle seems to exacerbate the defect, with a higher percentage of anucleate cells being observed with the [cir+] ulp1ES culture than with the others. These anucleate cells suggest cytokinesis may have occurred despite mitosis not having been properly executed. The [cir+] ulp1ES cells were often enlarged, had elongated buds, and displayed an apical chain-like budding pattern, phenotypes similar to those produced by overexpression of FLP in a [cir+] yeast strain (35).
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ES yeast was due to a delay in DNA replication or was at the G2/M boundary, the cells were analyzed by flow cytometry (Fig. 2). The [cir+] and [cir0] ULP1 cells both displayed the distribution expected for a population of unsynchronized wild-type cells, with approximately half the cells having the 2N DNA content of G2, 40% having the 1N DNA content of G1, and the remainder undergoing DNA replication (S). In contrast, a higher percentage of the population for both [cir0] and [cir+] ulp1ES yeast mutants had a 2N DNA content, consistent with the cell cycle delay at the G2/M boundary. This was most pronounced for the [cir+] ulp1ES yeast strain where almost no cells with a 1N DNA content were observed. In addition, a significant proportion of the [cir+] ulp1ES yeast cells had a DNA content higher than expected for a 2N chromosome complement.
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ES allele were found by Southern blotting to have a two- to fivefold amplification of the 2µm plasmid copy number relative to their parental ULP1 strain (Fig. 3 and data not shown). This amplification is similar to that previously observed with [cir+] nib1 mutants (17). Copy numbers for independent cultures of a given transformant varied. In addition, smooth colonies occasionally arose spontaneously in the [cir+] ulp1ES strain and were found by Southern blotting to have become [cir0] (data not shown). This clonal variability in plasmid copy number suggests a stochastic process in which some cells acquire either higher- or lower-than-average numbers of plasmid molecules at cellular division and that the frequency of these events may be increased in ulp1ES yeast.
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ES yeast results in clonal lethality of a subpopulation of the cells, observed as a nibbled colony morphology. Lethal sectors would not be produced if random cells in the colony arrested as a consequence of the mutation. Proliferation of neighboring cells would obscure their loss. Instead, cells that produce lethal sectors must continue to proliferate after they have become committed to death, pass their commitment on to their progeny, and then die during a later cell division. To test this hypothesis, individual [cir+] ulp1ES and ULP1 cells were assessed by a pedigree assay for their colony-forming ability (Table 3). As expected, all cells in the completed [cir+] ULP1 pedigrees produced normal large smooth colonies. Similarly, for over half of all pedigrees completed for the [cir+] ulp1ES mutant, all cells within the pedigree produced large but (in this case) variably nibbled colonies. In contrast, for about one-quarter of the completed [cir+] ulp1ES mutant pedigrees, all cells produced microcolonies of <100 cells. In the latter pedigrees, the results suggest that the initial mother cell had already become committed to cease proliferation and that daughter and the granddaughter cells inherited the commitment. Commitment might reflect an elevated plasmid copy number in these mother cells.
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ES mutation on growth.
To further investigate the basis of the ulp1ES defect, we compared the rate of growth of [cir0] and [cir+] ulp1ES strains to ULP1 yeast in liquid culture (Fig. 4). Both the [cir0] and [cir+] ulp1ES strains underwent a diauxic shift and entered stationary phase at a lower cell density than their respective ULP1 strains. Since the average size of a ulp1ES cell is greater than that of a ULP1 cell (Fig. 2), the lower cell density at diauxic shift may result from the larger mutant cells, exhausting the glucose at a lower cell density, irrespective of their plasmid content. In addition to an earlier entry into stationary phase, the [cir+] ulp1ES strain grew significantly slower during the logarithmic phase than either the [cir0] or [cir+] ULP1 strains or the [cir0] ulp1ES mutant strain. This growth difference is consistent with the [cir+] ulp1ES strain having a colony morphology that differs from that of the others. The reduced rate of logarithmic growth in the [cir+] ulp1ES yeast may represent the production of cells that constitute the lethal sectors in colonies formed by these strains.
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ES yeast cells obtained from a nibbled colony results in a variety of different colony sizes ranging from large normal-sized colonies to microcolonies of <100 cells and colony morphologies ranging from smooth to extensively nibbled (Fig. 5A). Cell size and shape are also more variable for the [cir+] ulp1ES yeast, even for cells within a single colony (Fig. 5B). To determine whether the different-sized and shaped colonies observed with [cir+] ulp1ES yeast reflected differences in plasmid levels within the cells of the colony, a semiquantitative PCR approach was undertaken (Table 4). For the DNA isolated from nibbled ulp1ES mutant colonies, 2µm plasmid levels showed a trend of increasing with decreasing colony size relative to the level of 2µm plasmid in a ULP1 strain. The smooth ulp1ES mutant colonies had only a twofold increase in 2µm plasmid levels. This less-dramatic increase in plasmid copy number might explain the loss of the nibbled colony phenotype. The correlation between the increase in 2µm plasmid levels and the loss of proliferative capacity, as reflected in reduced colony size, supports the hypothesis that elevated plasmid levels lead to cell death in ulp1ES yeast. Consistent with this hypothesis is the observation that [cir0] ulp1ES yeast does not produce nibbled colonies or microcolonies.
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ES yeast.
The Rep proteins have been shown to colocalize with the 2µm circle in the nucleus (48). To determine whether this localization was affected by the ulp1ES mutation, GFP-tagged versions of Rep1 (Fig. 7A) and Rep2 (Fig. 7B) were expressed in yeast. Both Rep fusion proteins colocalized with the DAPI-stained chromosomal DNA in the ulp1ES mutant cells. The nuclear distribution of the plasmid in the mutant did not significantly differ from that observed with ULP1 cells, indicating that the increase in plasmid copy number in the ulp1ES mutant was not due to altered localization of Rep1 and Rep2.
Plasmid segregation in ulp1ES yeast.
To determine whether 2µm plasmid distribution was affected by the ulp1ES mutation, a 2µm-derived reporter plasmid, pSV5, containing multiple lac operator sequences was visualized in cells expressing a GFP-LacI repressor fusion protein (48). The fluorescently tagged 2µm plasmids appeared as a small number of discrete foci colocalized with the DAPI-stained chromosomal DNA in both [cir+] ulp1ES and [cir+] ULP1 cells (Fig. 7C), indicating that plasmid localization within the nucleus did not require wild-type ULP1 function. Although the clustering of the plasmid in foci within the nucleus was unaffected, GFP-LacI fluorescence was not equally intense in some mother and daughter ulp1ES mutant cells, suggesting that the 2µm-derived reporter plasmid was not equally partitioned during mitosis (Fig. 7C). To assess this difference, cells with large buds having two distinct DAPI-stained masses, consistent with the mother cell having completed mitosis, were scored for their relative intensity of GFP-LacI repressor fluorescence (Table 6). Complete missegregation of the 2µm-derived reporter plasmid was observed with 6% of [cir+] ulp1ES mutant cells and not observed with [cir+] ULP1 cells, suggesting that the mutation in ULP1 impaired 2µm circle plasmid segregation.
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ES cell, the fluorescently tagged plasmids took >12 min to move from mother to daughter cell, and in some cells, all fluorescence was delivered to and retained in the daughter cell, consistent with complete missegregation of the plasmid in these mitotic divisions. The longer time taken for plasmid segregation in the [cir+] ulp1ES mutant is consistent with the G2/M lag observed for these mutant cells by flow cytometry (Fig. 2). The direction of plasmid missegregation contrasts with the maternal bias reported for plasmids that lack a functional partitioning system (6). However, more plasmid missegregation events need to be captured by time-lapse microscopy to establish whether missegregation is random or biased toward the daughter cell in the ulp1ES mutant. |
DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ES allele retained the 3' end of the ORF encoding the conserved protease domain (23) (33) and part of the noncatalytic regulatory domain required for retention of Ulp1 at nuclear pores (37). The remarkable similarity between the phenotypes conferred by this deletion allele and by nib1, a mutation that alters a single amino acid within the conserved carboxy-terminal protease domain, suggests that the ulp1ES allele expresses a truncated protein with protease activity sufficient for viability, but insufficient for normal plasmid maintenance. Both alleles may decrease the availability of free mature Smt3, either by impairing the proteolytic activity or by reducing expression of Ulp1. The loss of Ulp1 processing activity would reduce the level of some Smt3 conjugates, while other targets that are normally deconjugated by Ulp1 could accumulate (23, 25). The altered levels of these modified target proteins may be responsible for the phenotype of elevated 2µm circle plasmid levels common to both mutant ulp1 alleles. Although not specifically indicated, yeast strains used in previous experiments where either the ULP1 or UBC9 genes were disrupted or deleted (23, 45) would, like most strains of Saccharomyces cerevisae, probably be [cir+]. Altered levels of the 2µm circle may occur in these mutant yeasts. The distinct domains of nuclear staining with propidium iodide, observed when yeasts carrying temperature-sensitive alleles of the ULP1 gene were shifted 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-deconjugating protease, Ulp2 (24), or simultaneous deletion of MLP1 and MLP2, two genes encoding myosin-like proteins required for nuclear pore localization and stabilization of Ulp1 (52), also results in abnormal cell morphology, temperature-sensitive growth, and formation of nibbled colonies in [cir+] yeast, phenotypes remarkably similar to the 2µm circle-associated phenotypes observed with the ulp1ES mutant yeast. Accumulation of the 2µm circle in these mutant cells may also occur in response to loss or mislocalization of Smt3-deconjugating activity.
The lethality observed with [cir+] ulp1ES mutant yeast correlates with elevated plasmid copy numbers and may reflect titration of a host protein critical for progression through mitosis. Increased plasmid levels in the ulp1 mutant could arise from enhanced Flp recombinase-mediated amplification or from plasmid missegregation. Although plasmid missegregation seems more likely in light of the observed missegregation of the fluorescently tagged 2µm reporter plasmid, it is possible that altered Flp activity is the primary defect and that plasmid missegregation is a secondary response to elevated plasmid copy number. Altered Smt3 modification could stabilize the Flp protein or reduce Rep protein-mediated repression of FLP gene expression. In the alternative model, altered levels of a Smt3-modified protein(s) common to the pathways of both chromosome and plasmid segregation could be responsible for the G2/M cell cycle delay in the [cir0] ulp1ES mutant yeast and the missegregation of the multicopy plasmid observed with some [cir+] ulp1ES mutant cells. The higher-than-normal number of plasmids received by some cells could further impair plasmid segregation in subsequent divisions due to increased competition for limiting Smt3-conjugates. In cells that received less plasmid, the Flp recombinase would restore the normal plasmid copy number, the result being a continuous increase in plasmid levels in the culture.
Smt3 modification has been implicated in regulating chromosome segregation. The SMT3 gene was originally cloned on the basis of its ability, when overexpressed, to suppress the effects of mutation in Mif2, the yeast homolog of mammalian CENP-C, a kinetochore protein required for chromosome segregation and mitotic spindle integrity (31, 32). CENP-C may itself be SUMO modified (13). Smt3 modification may also participate in chromosome segregation by contributing to cohesion between chromosomes. Mcd1 and Smc1, two components of cohesin, a complex enriched at chromosomal centromeres that holds sister chromatids together until the onset of anaphase, are Smt3 modified (4, 22, 50). In a ulp2 mutant, centromeric cohesion is impaired in response to altered Smt3 modification of topoisomerase II (2), and a similar defect might be produced by loss of Ulp1 function. Since SUMO modification targets RanGAP1 to the spindle apparatus during mitosis (28), it is tempting to speculate that centromeric cohesin components and the 2µm circle use a similar approach, with Smt3/SUMO modification mediating attachment to the host spindle and conferring efficient segregation. In support of this hypothesis, the mitotic spindle has recently been shown to promote recruitment of the cohesin complex to the 2µm circle STB-partitioning locus (29).
The colocalization of the Ulp1 protease with the 2µm circle-encoded Rep proteins and the two-hybrid interaction of both Rep1 and Rep2 with Smt3 suggested the plasmid proteins might be targets of Smt3 conjugation or interact with Smt3-modified proteins. Evidence for Rep2, Flp, and possibly Rep1 being Smt3 modified has recently been obtained (E. Johnson, personal communication). The Rep proteins are essential for normal plasmid segregation at cytokinesis, are tightly associated with the 2µm circle DNA in a small number of discrete foci in live yeasts (48), and probably form a DNA-protein aggregate at the plasmid STB locus (6, 44). Efficient plasmid segregation involves recruitment of cohesin complexes to STB by the Rep proteins (30, 51). Cycling between Smt3-modified and unmodified forms of the plasmid proteins could allow their retention or release from a nuclear substructure in response to cell cycle signals, a process that may mediate both efficient plasmid segregation and copy number control. In the ulp1ES mutant, altered levels of Smt3-conjugated Rep proteins or cohesin components may affect plasmid association with the host spindle or impair separation of the 2µm plasmid-containing foci, structures that may colocalize with a yeast equivalent of the mammalian matrix-associated PML nuclear bodies (5, 11, 34). Identifying the targets of Ulp1 protease at the G2/M stage in the cell cycle may help reveal the mechanism by which a simple extrachromosomal DNA element parasitizes components of the sophisticated host cell cycle apparatus for its own stable, high-copy-number propagation.
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ACKNOWLEDGMENTS |
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This work was supported by an NSERC grant to M.J.D. and grants from the National Institutes of Health and the Robert Welch Foundation to M.J.
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FOOTNOTES |
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