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Molecular and Cellular Biology, May 2005, p. 4311-4320, Vol. 25, No. 10
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.10.4311-4320.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Misregulation of 2µm Circle Copy Number in a SUMO Pathway Mutant

Xiaole L. Chen, Alison Reindle, and Erica S. Johnson*

Department of Biochemistry and Molecular Pharmacology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

Received 6 October 2004/ Returned for modification 20 November 2004/ Accepted 2 February 2005


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Attachment of the ubiquitin-like protein SUMO to other proteins is an essential process in Saccharomyces cerevisiae. However, yeast mutants lacking the SUMO ligases Siz1 and Siz2/Nfi1 are viable, even though they show dramatically reduced levels of SUMO conjugation. This siz1{Delta} siz2{Delta} double mutant is cold sensitive and has an unusual phenotype in that it forms irregularly shaped colonies that contain sectors of wild-type-appearing cells as well as sectors of enlarged cells that are arrested in G2/M. We have found that these phenotypes result from misregulation of the copy number of the endogenous yeast plasmid, the 2µm circle. siz1{Delta} siz2{Delta} mutants have up to 40-fold-higher levels of 2µm than do wild-type strains. Furthermore, 2µm is responsible for the siz1{Delta} siz2{Delta} mutant's obvious growth defects, as siz1{Delta} siz2{Delta} [cir0] strains, which lack 2µm, are no longer heterogeneous and show growth characteristics similar to those of the wild type. Possible mechanisms for SUMO's effect on 2µm are suggested by the finding that both Flp1 recombinase and Rep2, two of the four proteins encoded by 2µm, are covalently modified by SUMO. Our data suggest that SUMO attachment negatively regulates Flp1 levels, which may partially account for the increased 2µm copy number in the siz1{Delta} siz2{Delta} strain.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The ubiquitin (Ub)-related protein SUMO (small ubiquitin-related modifier) functions by being covalently attached to other proteins as a posttranslational modification (12, 20, 28). Many proteins with diverse cellular functions are modified by SUMO, including at least 300 proteins in Saccharomyces cerevisiae (6, 13, 30, 41, 46). SUMO conjugation is essential for viability in S. cerevisiae, and sumoylation-defective conditional mutants arrest in the cell cycle at G2/M. However, it is not known which substrates are involved in the essential function(s) of SUMO. SUMO conjugation is also essential in most other eukaryotic cells.

SUMOs are a family of ~93- to 98-amino-acid proteins that are ~18% identical to Ub, a 76-residue modifier protein with several functions including targeting proteins for proteasome-dependent proteolysis (15, 31). Like Ub, SUMO is attached to lysine residues in substrate proteins through an amide bond linking the C terminus of SUMO to the {varepsilon}-amino group of the lysine residue. SUMO is often attached to the lysine in the sequence motif {Psi}KXE, where {Psi} is a hydrophobic residue. However, on some substrates, SUMO is attached to lysine residues in sequences that do not match this motif (20). SUMO conjugation can have a variety of different effects, including modulating protein-protein interactions, altering enzymatic activity, or blocking ubiquitylation of the substrate by competing for its ubiquitylation site lysine (12, 20, 28). SUMO does not directly target proteins for proteasome-dependent proteolysis.

SUMO is conjugated via a three-step enzyme pathway that first activates the SUMO C terminus and then modifies specific target proteins (12, 20, 28). This pathway consists of a heterodimeric SUMO-activating enzyme (E1) comprising Uba2 and Aos1, a SUMO-conjugating enzyme (E2) called Ubc9, and several SUMO ligases (E3s), which include the yeast proteins Siz1 and Siz2/Nfi1. Siz1 and Siz2 participate in sumoylation of partially distinct sets of substrates: some proteins can be sumoylated only by Siz1, others only by Siz2, and others can be sumoylated by either Siz protein (16, 22, 39; G. Bylebyl, A. Reindle, and E. S. Johnson, unpublished data). There is also some SIZ-independent sumoylation in yeast. Sumoylation is a reversible modification, and the SUMO-substrate bond can be cleaved by a family of SUMO-specific proteases, which includes the yeast proteins Ulp1 and Ulp2/Smt4. Ulp1 also processes the precursor form of SUMO to generate mature SUMO and therefore is required for both conjugation and deconjugation of SUMO. UBA2, AOS1, UBC9, and ULP1 are all essential genes, while ULP2, SIZ1, and SIZ2 are not. siz1{Delta} and siz2{Delta} single mutants grow quite well and do not have obvious phenotypes (22, 38, 39). However, the siz1{Delta} siz2{Delta} double mutant grows poorly at 30°C, forms irregularly shaped colonies, is cold sensitive, and contains many cells that are delayed in the cell cycle in the early stages of mitosis (22, 39). This result indicates that SIZ1 and SIZ2 have at least one overlapping function that contributes to these phenotypes.

We investigated the growth defects of the siz1{Delta} siz2{Delta} mutant and found that they involve the 2µm circle, an endogenous 6,318-bp plasmid that is found in virtually all laboratory and industrial strains of Saccharomyces (2). 2µm is a selfish DNA element whose only known activity is self propagation. 2µm encodes four proteins that promote efficient maintenance of the plasmid via two distinct mechanisms (2). One mechanism, which involves the 2µm-encoded proteins Rep1 and Rep2 as well as a cis element called STB or REP3, promotes accurate partitioning of 2µm between the mother and daughter cells at cytokinesis. In the absence of these factors, 2µm remains primarily in the mother cell. Rep1 and Rep2 bind STB and interact with the cellular cohesin complex, and these interactions are essential for accurate plasmid segregation (27, 44). The other mechanism enables 2µm to be amplified if its copy number drops. This involves a third 2µm-encoded protein, Flp1 recombinase, and two inverted repeat sequences, called FRT, that are present on opposite sides of the 2µm plasmid (2). Flp1 catalyzes intramolecular recombination between the two FRT sites, thereby inverting one side of the plasmid with respect to the other. 2µm has a single origin of replication, which fires once per S phase, but it is thought that Flp1 activity allows a single 2µm template to be copied more than once per S phase: if the Flp1-mediated intramolecular recombination takes place during S phase between the converging replication forks, it produces a replication intermediate in which both replication forks go in the same direction, chasing each other around the circle, producing multiple copies of 2µm DNA until the reaction is reversed and the structure resolved (2, 11). Expression of FLP1 is controlled by a mechanism involving the other three 2µm-encoded proteins (Rep1, Rep2, and Raf1), such that FLP1 expression is increased when 2µm levels are low and repressed when 2µm levels are high (2, 29, 32). This provides a feedback mechanism that promotes amplification of 2µm specifically when copy numbers drop.

2µm is generally considered to be an innocuous passenger in yeast, with only a 1.5 to 3% decrease in growth rate in a strain that contains the plasmid relative to an isogenic strain that does not (26). However, more than 20 years ago an allele called nibbled (nib) was described in which elevated levels of 2µm resulted in irregularly shaped colonies, cold sensitivity, and cell cycle delay (18, 19). The gene responsible for this phenotype was not identified. This result suggests that yeast has an "antiviral" mechanism for preventing 2µm from accumulating to such high copy numbers that it interferes with growth of the host cell.

We have found that the siz1{Delta} siz2{Delta} mutant, like the nib strain, has elevated levels of 2µm and that 2µm is involved in the major growth defects of this strain. We investigated possible molecular mechanisms for this misregulation of 2µm and found that both Rep2 and Flp1 are modified by SUMO, suggesting that SUMO may participate in 2µm copy number control by directly targeting plasmid-encoded proteins.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Media and genetic techniques. Standard techniques were used (1). Rich yeast medium containing 2% glucose (YPD) and synthetic yeast medium were prepared as previously described (36). {alpha}-Factor arrests were done with 10 µM {alpha}-factor (Sigma) for 3 h.

Plasmids and yeast strain constructions. S. cerevisiae strains used are listed in Table 1. All strains are derivatives of JD51 (8) except YWO1 and the ubc9 and ulp1 temperature-sensitive (ts) mutants, which are derived from DF5 (10). Deletions and epitope tags were made in the chromosomal copies of genes using the products of assembly PCRs as previously described (21). Construction details and oligonucleotide sequences are available on request. [cir0] strains were constructed by taking advantage of the fact that rsc2{Delta} mutants lose 2µm at a high rate (42). An rsc2{Delta}::kanR mutant was constructed in the JD51 background, cultured through three 1,000-fold dilutions, plated, and screened for [cir0] isolates by colony PCR (42). This rsc2{Delta} [cir0] strain was crossed to a siz1{Delta} siz2{Delta} rad9{Delta} [cir0] strain (EJY339), which we had obtained unintentionally when we attempted to construct this triple mutant in a [cir+] strain. A segregant containing none of the markers was selected, and this wild-type (wt) [cir0] strain was then mated to a siz1{Delta} siz2{Delta} [cir0] strain that had been made by crossing EJY339 to a siz1{Delta} siz2{Delta} CDC3-HA strain that we had also shown to be [cir0]. The resulting siz1{Delta}/SIZ1 siz2{Delta}/SIZ2 [cir0] diploid strain (EJY340) sporulated well, and all spores germinated and grew to produce colonies that were homogenous and grew well at all temperatures. Strains EJY341 to EJY344 were all derived from this diploid strain. Strains containing variants of 2µm were made from these [cir0] strains by transformation as described below.


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  		TABLE 1. S. cerevisiae strains

 
C-terminally tagged versions of 2µm-derived proteins were expressed from modified versions of the endogenous 2µm circle that did not contain an Escherichia coli origin or drug resistance marker. These were constructed by a PCR-based approach similar to the one used for adding epitope tags to chromosomal genes (21). Briefly, a PCR product containing ~500 bp of the 3' end of the desired open reading frame, followed by the tag, a STOP codon, a HIS3 or URA3 marker, and then the 3'-flanking region of the gene, was made by assembly PCR. This results in an insertion at the 3' end of the gene being tagged and should not affect the function of neighboring genes. The sequence of the hemagglutinin (HA) tag was GYPYDVPDYAAFL and that of the HA-His8 tag was GYPYDVPDYAAFLHHHHHHHH. The K375R and Y343F versions of FLP1 were made by similar approaches. Oligonucleotide sequences are available on request. These products were transformed into wild-type [cir+] yeast, and His+ transformants were selected. These were screened by PCR and immunoblotting for the presence of the tag and marker in the correct location. Genomic DNA (17) from these strains was then transformed into [cir0] versions of strains of the desired genotypes, followed by selection for His+ colonies. To make sure that the resulting transformants contained only the modified version of 2µm, colonies were screened by PCR using primers that flanked the marker in the tagged, marked plasmid (42). Colonies that contained the band diagnostic for the modified 2µm and lacking the band for unmodified 2µm were retained for further use. Primers used were 5'-GCCGTGGCCAGGACAACG-3' and 5'-GTAGCTCGTTACAGTCCG-3' for FLP1, 5'-GCCAGAGGATGGCGAACC-3' and 5'-GCTCGCGTTGCATTTTCG-3' for REP1, 5'-CTGGCGGCAGAGAATCGT-3' and 5'-CACTGTTCACGTCGCACC-3' for REP2, and 5'-GCACTTCTACAATGGCTG-3' and 5'-GCTTTCGCGTTGCATTTC-3' for RAF1. The wild-type and K375R versions of FLP1 were both amplified by PCR and sequenced. For the other genes, multiple independent clones were isolated and shown to have similar characteristics.

Antibodies and immunoblot analyses. Yeast whole-cell lysates were prepared by lysis in NaOH (43) and subjected to immunoblotting, followed by chemiluminescent detection (21). Antibodies were an affinity-purified rabbit polyclonal antibody (Ab) against Smt3 (SUMO) (21), the 16B12 monoclonal Ab against the HA epitope (Covance Research Products), and a goat polyclonal Ab against Rad9 (yC-20) (Santa Cruz Biotechnology). HA and His8-tagged proteins were purified from yeast by Ni-nitrilotriacetic acid (NTA) affinity chromatography (21, 41). Strains containing tagged 2µm genes marked with HIS3 were grown overnight in synthetic yeast-His medium and then diluted into 50 ml of YPD and grown to an optical density at 600 nm of ~1.0. Cells were collected by centrifugation, frozen in liquid N2, and lysed by the addition of 500 µl of 1.85 N NaOH and 7.4% ß-mercaptoethanol on ice for 10 min. Five hundred microliters of 50% trichloroacetic acid was added, and incubation on ice was continued for 10 min. The pellet was collected by microcentrifugation and washed twice by resuspension in ice-cold acetone. After removal of the acetone, the pellet was resuspended in 1 ml of 6 M guanidine-HCl, 50 mM NaPO4, 10 mM Tris (pH 8.0). All subsequent steps were done at room temperature. The pH was readjusted to ~8.0 by the addition of ~10 µl of 2 M Tris base, and 10 µl of 2 M N-ethylmaleimide in isopropanol was added. After the suspension was incubated for 20 min with rotation, the debris was removed by microcentrifugation for 10 min. Supernatants containing equal amounts of protein (assayed using Coomassie Plus; Pierce) were diluted to the same volume (1 ml) and supplemented to 30 mM imidazole. Ni-NTA agarose (~15 µl; QIAGEN) was then added, followed by incubation with rotation for 2 to 16 h. The supernatant was removed, and the Ni-NTA agarose was washed four times with 8 M urea, 50 mM NaPO4, 10 mM Tris, and 20 mM imidazole (pH 8.0). After the last wash, the beads were resuspended in 40 µl of Laemmli loading buffer and boiled, and 5 to 10 µl was loaded per well. Photographs of immunoblots were made using chemiluminescent detection (Supersignal; Pierce), but signals of bands were quantified with secondary antibodies coupled to fluorescent dyes IRDye 800 (Rockland Immunochemicals) and Alexa Fluor 680 (Molecular Probes) with an Odyssey infrared imaging system (LiCor Bioscience). Signals were approximately linear in the range where these bands were measured, but for some of the sumoylated species and for unmodified Flp1 in wild-type cells, the signal was not dramatically higher than background (sometimes only two- to threefold), so variations in the background could have substantial effects on the measurements.

Analysis of 2µm levels. The presence of 2µm was assessed by colony PCR as previously described (42) using the above primers from the 3' end of REP1. All of the primers listed above are effective in determining whether 2µm is present by colony PCR.

For quantitative real-time PCR (qPCR), yeast DNA was isolated using glass beads and phenol-chloroform (17). Levels of 2µm were measured using a sequence from the Y' subtelomeric element as the reference. Primers for measuring 2µm DNA were 5'-CACAAGATAGTACCGCAAAACGA-3' and 5'-CACCTTTGCTGCTTTTCCTTAATT-3', which amplify a 65-bp region between FLP1 and REP2. The Y'-derived primers were 5'-ACAATGGCCTTCGACTCTGGTTC and 5'-ATCACAGCCCGAAGAAGCACT-3'. These two amplicons had virtually identical amplification efficiencies and gave the same relative values over a 100-fold dilution of the template DNA. qPCR was carried out with an Opticon System DNA engine (MJ Research) using the DyNAmo HS SYBR green qPCR kit (MJ Research). Each reaction contained 15 µl of SYBR Green Master Mix, optimized concentrations of one of the primer sets (0.9 µM for each 2µm primer and 0.6 µM for each reference primer), 0.2 to 20 ng genomic DNA, and distilled H2O to a 30-µl final volume. PCR conditions were as follows: 1 cycle at 95°C for 14 min; and 40 cycles, each consisting of 95°C for 15 s, 60°C for 20 s, and 72°C for 30 s, followed by plate reading. The cycle number for the PCR product to reach preset threshold (CT number) was determined for three to six replicates for each DNA sample. The fold change of the 2µm number compared to that of wild-type yeast DNA was calculated by 2–{Delta}{Delta}CT methods (25). Values were compared by the Student t test.

Southern blot hybridization was performed as previously described (1). Lanes were loaded with 0.5 µg of yeast DNA, prepared as previously described (17), and digested with PstI. The probe consisted of a 2,246-bp ScaI-PstI fragment containing 2µm sequences from pRS426 (5). The probe was labeled using the DIG High Prime DNA labeling kit (Roche Applied Science), and the signal was detected according to the manufacturer's instructions.

Microscopy. Cells were stained with 1 µg/ml 4',6-diamidino-2-phenylindole (DAPI) after fixation in 90% ethanol. DAPI-stained and differential interference contrast micrographs were taken using a 63x oil objective on a Leica DM. RXA microscope with a Cool SNAP fx digital camera (Roper Scientific) and IP Lab software (Scanalytics). Photographs of yeast colonies were taken using a Nikon tetrad dissecting microscope with a 10x objective and 15x ocular by holding an Olympus Stylus 300 digital camera at 3x zoom to the ocular of the microscope.

Plasmid loss assays. To measure the loss rate of HIS3-marked versions of 2µm, single colonies from plates lacking His were grown for 15 to 25 generations in YPD, and the fraction of cells lacking the plasmid before and after growth in YPD was determined by plating ~300 cells on a YPD plate, allowing colonies to grow, and replica plating onto a plate lacking His to test for the plasmid. Loss rate was calculated as previously described (7). For Flp1-Y343F-containing URA3-marked plasmids, single colonies from plates lacking uracil were diluted and plated directly onto YPD plates, followed by replica plating onto plates lacking uracil. The results are the percentages of cells that have lost the plasmid while growing under selective conditions, which should represent the loss rate, assuming minimal cross-feeding within the colony.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
siz1{Delta} siz2{Delta} mutants exist in different heritable states. A siz1{Delta} siz2{Delta} double mutant formed colonies of varied size and cell morphology when grown at 30°C (Fig. 1) (22). Some colonies were near-wild-type sized and contained mostly small, normal-looking cells, but they also had some sectors of enlarged poorly growing cells, giving the colonies an irregular shape. Other colonies consisted entirely of the enlarged, poorly growing cells. While these often grew to contain several hundred cells, they usually could not be restreaked to form viable colonies. This effect depends on temperature: at 36°C, the highest temperature where the parental strain can grow, the double mutant is virtually indistinguishable from the wild type in its growth rate; whereas at 20°C, most cells are of the poorly growing type, and the strain is virtually inviable (22). This pattern of growth suggested that these mutant cells can grow in at least two different "states," both of which are heritable: cells in one state grow well and give rise to daughter cells that mostly grow well, whereas cells in the other state grow poorly and give rise to daughter cells that also grow poorly. The evidence also suggested that at certain rates, cells in either state could generate cells in the other state. For example, the healthy colonies contained sectors of poorly growing cells, suggesting that the cell that initiated the colony was of the rapidly growing type but that slowly growing cells were occasionally generated. The evidence for conversion in the other direction was that siz1{Delta} siz2{Delta} double mutants derived from tetrad segregation initially formed colonies consisting exclusively of enlarged, slow-growing cells, but eventually all spore-derived colonies generated the faster-growing, more normal-looking cells (data not shown).



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  		FIG. 1. siz1{Delta} siz2{Delta} strains form irregularly shaped colonies containing enlarged cells. Wild-type (left) or siz1{Delta} siz2{Delta} (right) cells were streaked on YPD plates and incubated at 30°C for 1 day (bottom) or 3 days (top). Top panels show visible colonies, while images in bottom panels were taken with a tetrad dissecting microscope (see Materials and Methods).

 
siz1{Delta} siz2{Delta} mutants have high 2µm copy numbers. It was pointed out to us that irregular colony shape, cold sensitivity, and G2/M arrest are characteristics of strains containing the nibbled (nib) allele, which was originally identified in a strain of Saccharomyces carlsbergensis (18, 19). These phenotypes of nib are associated with a high copy number of the 2µm circle. Consequently, we asked whether 2µm copy number was altered in siz mutants. siz1{Delta} and siz2{Delta} single mutants had 2µm copy numbers similar to that of the wt, but siz1{Delta} siz2{Delta} double mutant isolates had 7- to 40-fold-higher levels, as measured by quantitative PCR (Fig. 2A and B). 2µm levels varied substantially from isolate to isolate, which was not surprising, because these cultures were very heterogeneous in their growth rates. siz1{Delta} siz2{Delta} strains seemed to adapt as they were maintained in culture so that they grew better and had lower 2µm copy number levels. The isolate shown in Fig. 2A, lane 6, grew notably better than the others analyzed in the experiment shown in this figure (data not shown).



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  		FIG. 2. siz1{Delta} siz2{Delta} strains contain elevated levels of the 2µm circle. (A) Genomic DNA was isolated from cultures of the indicated strains grown in YPD at 30°C. Equal amounts of DNA were digested with PstI and analyzed by Southern blotting. The four siz1{Delta} siz2{Delta} lanes represent four different isolates of the same strain (EJY326). Numbers under the lanes represent the relative amounts of 2µm DNA in each of these DNA samples as determined by qPCR (see Materials and Methods). Levels were normalized relative to that of the wild type. (B) Single large round colonies of wt (JD52) or siz1{Delta} siz2{Delta} (EJY326) strains that had been grown on YPD plates at 36°C were split and restreaked on three different YPD plates and incubated at 36, 30, or 20°C for 2 days. Genomic DNA was isolated from the resulting cells and analyzed by qPCR (see Materials and Methods). Each of the grouped sets of results represents cells from the same original colony grown at each of the three temperatures. Error bars reflect the difference between replicate PCRs using the same DNA sample.

 
We also tested whether the cold sensitivity of siz1{Delta} siz2{Delta} correlated with differences in 2µm copy numbers at different temperatures. Four large round siz1{Delta} siz2{Delta} colonies growing at 36°C were each split and restreaked on YPD plates and grown for 2 days at 20°C, 30°C, and 36°C. Cells were grown on plates to prevent rapidly growing lineages from taking over the culture. 2µm copy numbers were then measured (Fig. 2B). Cells grown at 36°C contained 2- to 5-fold-higher levels of 2µm than wt cells, but cells grown at 30°C or 20°C contained 15- to 40-fold-higher levels. There was no significant difference in copy number between cells grown at 20°C and 30°C, even though the strain grew better at 30°C than at 20°C. Thus, 2µm levels partially correlated with growth rate, consistent with the idea that the higher the 2µm copy number, the slower the growth rate. There may be some other factor that also contributes to slow growth at 20°C.

2µm is responsible for major growth defects of siz1{Delta} siz2{Delta} mutants. To test whether absence of 2µm can suppress the cold sensitivity, heterogeneity, and abnormal colony and cell morphology of the siz1{Delta} siz2{Delta} strain, we constructed a diploid [cir0] strain that was heterozygous for both siz1{Delta} and siz2{Delta} (see Materials and Methods). This diploid strain sporulated like the wild type; all spores derived from this strain, including siz1{Delta} siz2{Delta} segregants, germinated and grew to produce colonies that were homogenous, grew well at all temperatures and were virtually indistinguishable at a superficial level from those of the wild type (Fig. 3A). If we reintroduced 2µm into the haploid siz1{Delta} siz2{Delta} [cir0] strain by transforming it with genomic DNA from a yeast strain where a marker had been introduced into 2µm, the transformed colonies were cold sensitive, showed the nibbled appearance, and contained enlarged arrested cells (Fig. 3A and data not shown). These results demonstrate that absence of 2µm suppresses siz1{Delta} siz2{Delta} phenotypes. Cultures of the siz1{Delta} siz2{Delta} [cir0] strain also contained many fewer enlarged cells with undivided nuclei than did siz1{Delta} siz2{Delta} [cir+] cultures (Fig. 3B; Table 2). However, the siz1{Delta} siz2{Delta} [cir0] strain did contain slightly more of these arrested cells than did a wt culture (Table 2), suggesting that this defect is not entirely dependent on 2µm. Table 2 shows the percentages of G2/M-phase (undivided nucleus) and M-phase (divided nucleus) cells present in cultures that had been exposed to {alpha}-factor, which causes cells to arrest in G1. {alpha}-Factor was used to facilitate the counting of cells arrested at other points in the cell cycle. We do not believe that siz mutations or 2µm affected the response to {alpha}-factor itself, because most cells arrested and formed mating projections like wt and because we also saw arrested cells in log-phase cultures.



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  		FIG. 3. Many phenotypes of the siz1{Delta} siz2{Delta} strain are suppressed by elimination of 2µm. (A) Cells of the indicated genotypes were streaked on a YPD plate and incubated at 20°C for 4 days. (B) Microscopy of wt (top), siz1{Delta} siz2{Delta} [cir+] (middle), or siz1{Delta} siz2{Delta} [cir0] (bottom) cells grown at 30°C using differential interference contrast (DIC) Nomarski optics (left) or DAPI staining (right). Bar, 5 µm. (C) Whole-cell lysates from cultures of the indicated genotypes were analyzed by SDS-PAGE and immunoblotting with an Ab against Rad9. Bands corresponding to Rad9 are indicated.

 

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  		TABLE 2. Percentage of cells at various stages of the cell cycle in {alpha}-factor-arrested culturesa

 
We also observed that siz1{Delta} siz2{Delta} [cir+] strains contained high levels of slow-migrating forms of Rad9 (Fig. 3C), which likely represent the hyperphosphorylated forms that are generated upon DNA damage (9, 40). Levels of these forms of Rad9 were dramatically reduced in the siz1{Delta} siz2{Delta} [cir0] strain and were restored by reintroducing 2µm (Fig. 3C). This result suggests that some feature of 2µm in these cells activates the Rad9-dependent checkpoint. However, the siz1{Delta} siz2{Delta} [cir0] strain contained reproducibly higher levels of these forms than did the wild type, suggesting that there is some 2µm-independent activation of the Rad9-dependent checkpoint in this mutant.

The strong suppression of the siz1{Delta} siz2{Delta} strain's phenotypes by the loss of 2µm prompted us to test whether the absence of 2µm would also suppress the lethality of a strain entirely lacking SUMO, which in S. cerevisiae is encoded by the SMT3 gene. We sporulated a siz1{Delta}/SIZ1 siz2{Delta}/SIZ2 smt3{Delta}/SMT3 [cir0] diploid and found that all smt3{Delta} segregants arrested permanently after only three to four cell divisions, indicating that the lethal phenotype of the smt3{Delta} strain does not result from a defect in 2µm copy number control. We also assayed a number of other previously isolated SUMO pathway mutants for 2µm and found that our isolates of ts mutants in ubc9 (ubc9-P69S) (35), uba2 (uba2-ts10) (23, 33) and ulp1 (ulp1-333) (24) were all [cir0], even though their parental strains were [cir+] (Fig. 4). This result suggests that either these strains have a high loss rate of 2µm, impose a selection for [cir0] derivatives, or both. Two different ulp2 mutants that we tested were both [cir+], and when we constructed ulp2{Delta} [cir0] cells, as described above for smt3{Delta}, the ulp2{Delta} [cir0] cells still had notable growth defects, indicating that the most conspicuous phenotypes of ulp2{Delta} cells are not dependent on 2µm (data not shown).



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  		FIG. 4. Isolates of many SUMO pathway mutants are [cir0]. Previously isolated SUMO pathway mutants were analyzed for the presence of 2µm DNA by colony PCR (see Materials and Methods), followed by agarose gel electrophoresis and ethidium bromide staining. The arrow indicates the band that is diagnostic for the presence of 2µm.

 
Flp1 and Rep2 are modified by SUMO. 2µm encodes four proteins, and one possible cause of the increased 2µm copy number in siz1{Delta} siz2{Delta} is that it results from insufficient sumoylation of one of these proteins. The other possibility is that under-sumoylation of a yeast host protein is involved. To examine sumoylation of the 2µm-encoded proteins, we constructed strains containing versions of endogenous full-length 2µm in which each of the four proteins was C-terminally tagged with HA and His8 tags. These strains contained only the tagged version of 2µm, with none of the original native plasmid (see Materials and Methods). While the plasmids expressing tagged Rep1 and Rep2 were not maintained as well as the Flp1- and Raf1-expressing plasmids (~1% loss per generation for tagged Rep1 and Rep2 versus 0.3% for Flp1 and 0.08% for Raf1) (see Materials and Methods), these loss rates are an order of magnitude lower than those seen when the plasmid segregation system is inactive (34, 42, 44). This suggested that tagged Rep1 and Rep2 were at least partially active. The Flp1-HA-His8 and the Flp1-K375R-HA-His8 discussed below were also active, as the plasmids containing them were present in both Flp1-convertible inversion isomers, even though the original transformants presumably contained only one isomer (data not shown). Also, a C-terminal tag has been shown previously not to affect Flp1 activity in vitro (4). However, as discussed below, none of these tagged versions of 2µm, which also contained a HIS3 marker, accumulated to as high copy numbers in a siz1{Delta} siz2{Delta} strain as did unmodified 2µm, suggesting that the marker and/or the tags interfered somewhat with 2µm biology. Also, we suspect that Rep1-HA-His8 was present at reduced levels relative to untagged Rep1, because similar plasmids with only the HA tag produced a much stronger signal on Western blotting with anti-HA. However, the His8 tag is essential for this experiment.

Tagged proteins were enriched by Ni2+ affinity chromatography, followed by immunoblotting with Abs against the HA epitope or against Smt3 (SUMO). This experiment demonstrated that both Flp1 and Rep2 were modified by SUMO, with three modified forms of Flp1 and at least three of Rep2 (Fig. 5A). (Unsumoylated Rep2 ran as two major bands, and there appeared to be two corresponding sumoylated forms with each of one, two, etc., SUMO moieties.) It is likely that these represent SUMO attachment at three different sites on Flp1 and Rep2. With a very long exposure, we could also detect a band that might be a sumoylated form of Rep1 (data not shown). Next, versions of 2µm containing tagged Flp1 and Rep2 were introduced into siz1{Delta}, siz2{Delta}, and siz1{Delta} siz2{Delta} strains, and the same experiment was performed (Fig. 5B). Several results were notable in the Flp1 experiment. One was that the fraction of Flp1 that was sumoylated was increased ~2 to 3-fold in the siz2{Delta} strain but decreased by about 95% in the siz1{Delta} siz2{Delta} mutant. Also, there was a 5- to 10-fold more Flp1 protein in the siz1{Delta} siz2{Delta} mutant than in the wild type. A priori, this increase in Flp1 levels could be either a cause or an effect of the high 2µm levels in this mutant. However, these tagged, marked versions of 2µm did not accumulate to as high copy numbers in the siz1{Delta} siz2{Delta} strain as did unmodified 2µm (data not shown; Fig. 5B), and analysis of a DNA sample taken from the same culture as the protein sample showed that the cells where Flp1 levels were almost 10-fold higher contained only about 2-fold more 2µm DNA (Fig. 5B). This result suggests that high levels of 2µm cannot fully account for the increase in Flp1 levels. In contrast to Flp1, the fraction of sumoylated Rep2 was only slightly lower (20 to 50%) in the siz1{Delta} siz2{Delta} double mutant than in the wild type. These results are consistent with a model where the siz1{Delta} siz2{Delta} mutant affects 2µm via an effect on Flp1.



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  		FIG. 5. Flp1 and Rep2 are modified by SUMO. (A) Lysates from cells containing versions of 2µm in which different 2µm-encoded proteins were tagged with the HA epitope and a His8 tag were subjected to Ni-NTA affinity chromatography (see Materials and Methods). Eluates were analyzed by SDS-PAGE and immunoblotting with Abs against HA (left) or Smt3 (right). Tagged proteins are indicated over the lanes. Bands corresponding to unmodified Rep1, Flp1, Rep2, and Raf1 are indicated. A band that cross-reacted with the anti-HA antibody is indicated by an asterisk. (B) Strains of the indicated genotypes containing 2µm with Flp1-HA-His8 (left panels) or Rep2-HA-His8 (right panels) were analyzed as in panel A using antibodies against HA (bottom panels) or Smt3 (top panels). Arrowheads indicate the unmodified proteins (there were two major bands of Rep2), and lines indicate SUMO-modified forms. Asterisks indicate bands that cross-reacted with the antibodies. Numbers under the lanes indicate the relative quantities of the unmodified Flp1 or Rep2 band, the monosumoylated band, and the amount of 2µm DNA in the same culture from which protein bands were quantified (see Materials and Methods). Quantities were normalized relative to the wild type. For Rep2, the first two sumoylated bands, which likely represent monosumoylation of the two different forms of Rep2, were pooled for protein quantification.

 
To test this, we wanted to mutate the SUMO attachment sites in Flp1. The fact that there were three apparent bands of sumoylated Flp suggests that Flp1 is sumoylated at three separate sites. However, Flp1 contains only one sequence that matches the SUMO attachment site consensus sequence, at Lys375 in the sequence LKDE. (Rep2 contains no consensus sequences.) We constructed a version of 2µm where HA-His8-tagged Flp1 contained a Lys-to-Arg mutation at this position and found that this dramatically reduced the levels of sumoylated Flp1 (Fig. 6A), suggesting that Lys375 is a major site of Flp1 sumoylation. The two remaining sumoylated forms in this mutant migrated more rapidly during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) than the corresponding wild-type forms did, suggesting that they were different from those seen in the wild type, possibly because they contained linkages at different sites. Furthermore, in the siz1{Delta} mutant especially, there were two bands of the approximate size of monosumoylated Flp1, which may represent forms that are each monosumoylated a different site. These results are consistent with a model where Lys375 is a major sumoylation site in Flp1 but where there are at least two other sites.



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  		FIG. 6. Lys375 of Flp1 is a major sumoylation site, and mutating it increases the amount of Flp1 protein. (A) Yeast strains of the indicated genotypes contained either native 2µm or 2µm with wild-type Flp1-HA-His8 or Flp1-K375R-HA-His8, as indicated. Analysis was as described in the legend to Fig. 5 with immunoblotting with anti-Smt3 (top) or anti-HA (bottom). Designations are as described in the legend to Fig. 5, except that sumoylated bands are designated with open circles placed on top of the image. Numbers under the lanes indicate the quantity of unsumoylated Flp1 relative to that in the strain with wild-type tagged Flp1. (B) Genomic DNA was isolated from cells of the indicated genotypes containing the indicated version of 2µm and analyzed by qPCR (see Materials and Methods). HH, HA-His8 tag. Quantities were normalized to the value in wt cells containing unmodified 2µm. Error bars represent variations between DNA preparations from three separate isolates of each strain. (C) Microscopy of colonies from wild-type strains containing HA-His8 versions of wild-type Flp1 (top) or K375R-Flp1 (bottom) that were grown for 2 days on a YPD plate at 20°C.

 
Like the siz1{Delta} siz2{Delta} mutant, the wild-type (SIZ1 SIZ2) strain containing Flp1-K375R had increased levels (~4-fold) of Flp1 (Fig. 6A), suggesting that deficient sumoylation of Flp1 may result in higher Flp1 levels. SIZ1 SIZ2 strains containing Flp1-K375R-HA-His8 also reproducibly had slightly higher levels of 2µm DNA than cells containing wild-type Flp1-HA-His8 (Fig. 6B). The difference was quite small, only ~2-fold, but significant, with a P value of <0.05. Similar results were also obtained using different plasmids that expressed Flp1-HA and Flp1-K375R-HA. This result is consistent with a model where deficient sumoylation of Flp1 contributes to increased 2µm levels. However, levels of 2µm in the SIZ1 SIZ2 Flp1-K375R-HA-His8 strain were significantly lower than in siz1{Delta} siz2{Delta} strains containing either wild-type or mutant Flp1. This result suggests that reduced sumoylation at Lys375 only partially accounts for the increased 2µm levels in siz1{Delta} siz2{Delta} strains. The difference between the siz1{Delta} siz2{Delta} strains containing wild-type versus mutant Flp1 was not significant. Consistent with a partial role for sumoylation of Flp1 Lys375 in maintaining normal 2µm levels, the SIZ1 SIZ2 strain containing Flp1-K375R-HA-His8 produced slightly more abnormal colonies when grown at 20°C than did a strain with wild-type Flp1-HA-His8 (Fig. 6C). In general, however, the Flp1-K375R-HA-His8 strain grew at approximately wild-type rates at all temperatures. To confirm that accumulation of 2µm in siz1 siz2 strains was indeed Flp1 dependent, we examined 2µm copy number and plasmid loss rates of a version of 2µm in which the active site Tyr-343 of Flp had been mutated to Phe. The copy number of this plasmid was the same in wt and siz1 siz2 strains (0.5 ± 0.1 for wt cells versus 0.6 ± 0.3 for siz1 siz2 cells, both values relative to the copy number of native 2µm in wt [JD52] cells), as was the plasmid loss rate (6% ± 6% for the wt versus 6% ± 3% for siz1 siz2). A similar experiment with a 2µm-based plasmid completely lacking Flp1 (pAS4) (34) gave similar results (data not shown). As would be expected, siz1 siz2 strains containing these plasmids did not contain high fractions of abnormal cells. In summary, these results show conclusively that the Flp1-K375R mutation increases Flp1 protein levels and suggest that deficient sumoylation of Lys375 may partially account for the effect of siz1{Delta} siz2{Delta} mutants on 2µm copy numbers.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We investigated the cold sensitivity, irregular colony shape, and cell cycle delay of siz1{Delta} siz2{Delta} mutants and found that all of these phenotypes are dependent on the endogenous 2µm circle plasmid. siz1{Delta} siz2{Delta} cultures contained up to 40-fold-higher levels of 2µm than the wild type, and this high copy number is likely to be responsible for the phenotypes. We also found that Flp1 and Rep2, two of the four proteins encoded by 2µm, were modified by SUMO. Flp1 sumoylation was dramatically reduced in the siz1{Delta} siz2{Delta} mutant, and this strain also contained significantly increased levels of Flp1 protein, suggesting that the SUMO pathway may negatively regulate Flp1. Additional support for this idea comes from the observation that mutating a major SUMO attachment site in Flp1 also increased Flp1 protein levels. Increased expression of FLP1 has been shown previously to increase 2µm levels (29, 32), raising the possibility that the high Flp1 levels may be partially responsible for the accumulation of 2µm in these strains.

While 2µm is responsible for the more conspicuous defects of the siz1{Delta} siz2{Delta} strain, 2µm copy number control is not the only function of SIZ1 and SIZ2. Siz1 and Siz2 are responsible for sumoylating many proteins, most of which presumably do not affect 2µm. For example, sumoylation of yeast PCNA requires SIZ1, and there are DNA repair defects associated with the loss of PCNA sumoylation (14, 16, 37). Furthermore, siz1{Delta} siz2{Delta} [cir0] strains contained slightly higher levels of highly phosphorylated Rad9 than did wt cells, suggesting that they contain damage in their chromosomal DNA.

Previous discussions of the nib phenotype have argued that nib could either be the cause of high 2µm levels or that the nib strains might merely be hypersensitive to higher than normal levels of 2µm (2, 18). Absence of SIZ1 and SIZ2 clearly caused the elevated 2µm copy number here. Entire cultures grown from siz1{Delta} siz2{Delta} [cir+] segregants, which were derived from heterozygous diploid strains with normal 2µm copy numbers, had dramatically higher copy numbers than wild-type segregants from the same tetrads. The cells in these colonies were uniformly sick within a few cell divisions of germination and only later developed sectors that grew better and had lower 2µm levels. The role of SIZ1 and SIZ2 in 2µm biology is, almost certainly, to promote SUMO conjugation; there is currently no evidence that SIZ1 and SIZ2 have any functions independent of SUMO conjugation, although the related mammalian PIAS proteins can have SUMO-independent effects (20). Furthermore, other mutants in the SUMO pathway also have 2µm-related defects. The original nib allele has been mapped to a region near RAD1 (19), which is very close to ULP1, a gene encoding a SUMO cleaving enzyme. Recently, it has been shown that nib is indeed an allele of ULP1 (M. Dobson, personal communication). Very recently, it has also been shown that a mutant that mislocalizes Ulp1 also accumulates 2µm (45). We also found that our isolates of ts mutants in ubc9, uba2, and ulp1 are all [cir0], although their parental strains are [cir+], suggesting that growth of these strains may have selected for the loss of 2µm.

What are the SUMO substrates involved in preventing accumulation of 2µm? Our results suggest that sumoylation of Flp1 may account at least partially for this role of the SUMO pathway. Flp1 sumoylation was at near or above wild-type levels in siz1{Delta} and siz2{Delta} single mutants but was reduced by about 95% in the double mutant. This result is consistent with the genetics of 2µm accumulation, where 2µm levels were about the same as in the wt in both single mutants but were dramatically increased in the double mutant. Furthermore, total amounts of Flp1 were increased 5 to 10 fold in the siz1{Delta} siz2{Delta} double mutant. This suggests a possible mechanism for how 2µm levels become elevated, as it has been shown that high levels of Flp1 are sufficient to increase 2µm copy number (29, 32). On the other hand, one could argue that reduced sumoylation of some other protein might cause the increase in 2µm levels and that the increased 2µm levels would cause the increase in Flp1. This idea is contrary to the model for feedback regulation of FLP1 expression and is also made less likely by our observation that a strain containing 10-fold-higher levels of Flp1 contained only ~2-fold-more 2µm than the wild type. Another argument that changes in Flp1 levels may be the cause rather than the effect is that the Flp1-K375R mutant, which eliminated a major sumoylation site in Flp1, also showed elevated levels of Flp1 protein. In this case, elevated Flp1 levels cannot be a secondary effect from deficient sumoylation of a different protein. Of course, this mutation could have effects on Flp1 other than just reducing sumoylation, but the fact that this mutation and the siz1{Delta} siz2{Delta} mutant had similar effects is consistent with a model where sumoylation of Flp1 at this site leads to a reduction in Flp1 protein levels. Lys375 is in a C-terminal domain of Flp1 that is not present in the related Cre recombinase (3), consistent with the possibility that this is a regulatory domain unique to Flp1.

However, reduced sumoylation of Flp1 at Lys375 does not account fully for the high levels of 2µm seen in siz1{Delta} siz2{Delta} mutants. Significantly higher levels of Flp-K375R-containing 2µm DNA were present in the siz1{Delta} siz2{Delta} strain than in the wild-type strain, indicating that SIZ1 and SIZ2 have other roles in 2µm control. This difference could reflect a requirement for SIZ1 and SIZ2 in attaching SUMO to Rep2 (or possibly Rep1), to host proteins or to other sites in Flp1. There was not a dramatic difference in sumoylation levels of Rep2 in the siz1{Delta} siz2{Delta} strain compared to the wild type, which might suggest that Rep2 is less likely to play a major role in the phenomenon. Sumoylation of the other sites in Flp1 was Siz dependent. Several yeast proteins involved in 2µm partitioning have recently been shown to be sumoylated, including the chromatin remodeling complex subunit Rsc2 and the cohesin subunits Smc1 and Scc1/Mcd1 (27, 41, 42, 44). It is not known to what extent sumoylation of these proteins is controlled by Siz1 and Siz2.

While this discussion has focused on how alterations in Flp1 could produce high levels of 2µm DNA, defects in plasmid partitioning could also lead to accumulation of 2µm. Unequal plasmid segregation would be followed by amplification in the progeny cell (most likely the daughter cell) that receives the lower level of plasmid. This would cause an overall accumulation of 2µm, as long as complete loss of the plasmid was infrequent. Flp1 would be required for 2µm accumulation under this model as well: 2µm copy numbers would not be expected to rise overall without Flp1-dependent amplification in the cells receiving fewer copies. Our results using Flp1-deficient versions of 2µm showed that 2µm is indeed amplified by a Flp1-dependent mechanism in siz1 siz2 strains. This is consistent with either the hyperactive Flp1 model or the defective partitioning model for accumulation of 2µm.

Further studies will be needed to resolve the many remaining questions about this system, including the identities of the other SUMO attachment sites in Flp1 and Rep2, the roles of other host proteins in controlling SUMO-dependent 2µm-related functions, and the mechanism by which 2µm amplification is triggered in some cells but not in others.


   ACKNOWLEDGMENTS
 
We are very grateful to Virginia Zakian for calling nibbled to our attention. We thank Michael King's lab for use of their microscope, Eric Wickstrom's lab for use of their real-time PCR system, and Adam Dicker's lab for use of their LiCor Odyssey, which was generously loaned by LiCor. We also thank Greg Cesarone, Qing Ren, and Bill Davidson for help using equipment. We thank Gwen Bylebyl for the initial HA-His8 tag construct and Aseem Gupta for technical assistance. We also thank Melanie Dobson for communicating results prior to publication, plasmids, and for comments on the manuscript and Marcus Shin for comments on the manuscript.

This work was supported by grants GM62268 from the National Institutes of Health and C0302 from the W. W. Smith Foundation.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biochemistry and Molecular Pharmacology, Thomas Jefferson University, 233 South 10th St., BLSB 231, Philadelphia, PA 19107. Phone: (215) 503-4616. Fax: (215) 503-5393. E-mail: Erica.Johnson{at}jefferson.edu. Back


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 Discussion
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Molecular and Cellular Biology, May 2005, p. 4311-4320, Vol. 25, No. 10
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.10.4311-4320.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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