INTRODUCTION

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The 2µm circle, a relatively small circular plasmid (6,318 bp) present in most common strains of Saccharomyces cerevisiae, has optimized a partitioning system and an amplification system that allow it to be propagated stably in a cell population at a copy number of approximately 60 to 100 per cell (reviewed in reference 2). Genetic analyses suggest that two plasmid-coded proteins, Rep1p and Rep2p, in conjunction with a cis-acting locus STB (also called REP3) contribute to the stability function (16, 17, 19, 23). One plausible mechanism for plasmid stability is that the interaction of Rep1p and Rep2p with the STB element serves to overcome the normal bias in plasmid segregation that tends to favor the mother cell over the daughter cell (22). The evidence for this suspected DNA-protein interaction is quite preliminary and rests almost entirely on the observation that urea-solubilized yeast extracts expressing Rep1p and Rep2p or [cir⁰] extracts supplemented exogenously with Rep1p and Rep2p can bind STB (14).

The need for plasmid amplification arises only if and when there is a decrease in copy number below the steady-state value. Normally, each plasmid molecule is replicated once, and only once, per cell cycle (35), and the daughter molecules are partitioned efficiently at cytokinesis (27). When there is a drop in copy number, the amplification system overrides the cell cycle restriction of a single round of plasmid replication during one S phase. Plasmid amplification is absolutely dependent on the 2µm circle Flp site-specific recombination system (33). A currently favored model for amplification proposes the recombinational inversion of a bidirectional replication fork and the resultant double-rolling-circle replication mode as the means for obtaining multiple replicas of the plasmid from a single initiation event (9-11, 26). The cessation of amplification would require a second recombination event that can restore the normal direction of fork movement. The time interval between the two successive recombination events would determine the degree of amplification.

How is the amplification system kept silent under normal steady-state growth conditions? And how is it rapidly commissioned into action at short notice? Biochemical answers to these fundamental question are sparse. On the basis primarily of genetic studies, it has been proposed that a regulatory complex containing Rep1p and Rep2p may provide an indirect readout of the copy number and, either at or above a critical concentration, may negatively control amplification by turning down expression of the FLP gene (24, 25, 28, 30). Recently, we have demonstrated self- and cross-interactions between Rep1p and Rep2p by immunoprecipitations of these proteins from mixed extracts of Escherichia coli cells that express them and by baiting assays with hybrid glutathione S-transferase (GST)-Rep proteins (1). Furthermore, these findings were corroborated by in vivo assays in yeast cells. We now present an analysis of the polypeptide regions in Rep1p and Rep2p responsible for their cellular localization as well as for their functional interactions. We also show here that an in vivo assay with the STB locus as a DNA bait fishes out three proteins: the Rep1 and Rep2 proteins, as well as a chromosomally encoded protein (the product of the YIL036W locus in the yeast genome bank). The chromosomal protein can bind to the STB element in vitro as well. Furthermore, its absence in vivo causes high instability of a 2µm circle-derived test plasmid. The potential implications of these findings in the benign parasitism of the 2µm circle plasmid are discussed here.

	MATERIALS AND METHODS

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Yeast strains. The following [cir⁰] and [cir⁺] yeast strains were used in this study: FVY2-6B (MAT leu2-3,112 ura3-52 his3-d1 [cir⁰]; Gal⁺), FVY889 (MAT ade2 leu2-3,112 ura3-52 his5-2 [cir⁰]; Gal⁺), FVY93154 (MATa leu2-3,112 ura3-52 trp1-289 ade2 [cir⁺]; Gal⁺), CCY666-1A (MATa ade2 his3-200 leu2-3,112 ura3-52 [cir⁺]; Gal⁺), CCY666-7B (MAT ade2 his3-200 leu2-3,112 ura3-52 [cir⁺]; Gal⁺), and MJY101 (CCY666-7B, shf1::URA3). The cellular localizations of green fluorescent protein (GFP) hybrids were done in strains FVY2 [cir⁰] and FVY93154 [cir⁺], respectively. Results were unchanged when FVY889 and CCY666-1A were used as the [cir⁰] host and the [cir⁺] host, respectively. In the Results section, only the data from FVY2 [cir⁰] are presented. The stabilities of the ADE2 containing test plasmids pSTB1 and pSTB2 (see below) were assessed in yeast strains FVY889 and CCY666-1A. The strain EGY48 (his3 trp1 ura3-52 leu2::pLEU2-LexAop6 [cir⁺]) used for the dihybrid assays was kindly provided by Roger Brent (8), and the strains used for the monohybrid assays were obtained from Clontech Laboratories (Palo Alto, Calif.). The dihybrid test was also performed in a [cir⁰] strain harboring the requisite markers. The outcomes were identical in the [cir⁰] and [cir⁺] strains. The host for the monohybrid assay, supplied by Clontech Laboratories, was a [cir⁺] strain. This strain was first crossed with a [cir⁰] partner, and a haploid [cir⁰] strain with the appropriate markers (ura3, his3, leu2, and trp1) was derived from the cross. The monohybrid analysis was done in this [cir⁰] derivative.

Plasmids. The pGFP-Rep plasmids, obtained by joining the 2µm circle REP1 and REP2 coding regions to the 3' end of the GFP coding region via a short in-frame adapter sequence, have been described previously (1). The cloning manipulations were done in the yeast GFP expression vector pTS408 (an ARS-CEN-URA3 plasmid obtained as a gift from the Botstein laboratory through Clarence Chan). In this plasmid, the GFP gene was controlled by the yeast GAL1-10 promoter. The deletions in the Rep portions of these plasmids were obtained by PCR with primers containing appropriate restriction enzyme sites to facilitate cloning. The nuclear localization signal (NLS) derived from the simian virus 40 (SV40) T antigen was inserted into some of these plasmids in the form of a synthetic DNA fragment encoding the heptapeptide (₂HN-PKKKRKV-COOH). The DNA sequence corresponding to this heptapeptide was the same as that in plasmid pGAD424 (from nucleotide positions 452 to 472), which was supplied by Clontech Laboratories.

Fluorescence microscopy of cells. The protocols for assaying the green fluorescence from GFP-Rep protein fusions and the blue fluorescence from DAPI (4',6-diamidino-2-phenylindole) complexed with DNA were as detailed by Ahn et al. (1).

Stability assays for plasmids pSTB1 and pSTB2. Stabilities of the pSTB1 and pSTB2 plasmids in a [cir⁰] host harboring pGFP-Rep or pGFP-Rep deletion plasmids were assayed as follows. Purified colonies of the plasmid-bearing strains (LEU2 and ADE2 markers on pSTB1 or pSTB2; URA3 on pGFP-Rep) were maintained on SD plates without leucine or uracil-glucose or on SD plates without leucine or uracil-galactose. Single colonies from the glucose and galactose master plates were spread out on yeast extract-peptone-dextrose (YEPD) and YEP-galactose plates, respectively, and grown for 3 days at 30°C. The red and white colonies on each plate were counted. Sectored colonies were grouped with the white colonies if the sector size was smaller than one-fourth the colony size, and with the red colonies if the sector size was larger. The plasmid stability index (SI) was then expressed as the ratio of the white colonies to the sum of the white plus red colonies multiplied by 100. The values of SI given here are for transfer of colonies from selective galactose plates to YEP-galactose plates (see Table 2).

Stability of the pSTB plasmid in an shf1::URA3 yeast strain. The test plasmid was pSTB, containing the 2µm plasmid origin, the STB locus, and the yeast ADE2 and LEU2 markers (1). However, it did not contain either REP1 or REP2. Plasmid stability was assayed simultaneously in the parent strain CCY666-7B (wild type for the SHF1 locus) and in its shf1::URA3 derivative (strain MJY101). Individual transformants purified on SD plates lacking leucine and uracil were grown in YEPD liquid medium for approximately 10 generations and plated on YEPD plates, and the colony color was screened after approximately 60 h of growth at 30°C.

Baiting assays with hybrid GST-Rep proteins. The expression of the GST-Rep hybrid proteins or the S-tagged Rep proteins in E. coli and the preparation of cell supernatants for the baiting assays followed the protocols detailed by Ahn et al. (1). Each baiting mixture contained 1.0 ml of the GST-Rep1p (or GST-Rep2p) plus 1.0 ml of the S-Rep2p (or S-Rep1p) supernatant.

Dihybrid and monohybrid assays in yeast cells. The dihybrid assays were carried out according to procedures described by Finley and Brent (8). The -galactosidase activities were assayed according to the method of Guarente (12). The monohybrid assays were done according to the protocols provided by Clontech Laboratories. An approximately 375-bp fragment from the 2µm plasmid spanning the STB locus was amplified by PCR and cloned upstream of the basal promoter of the HIS3 reporter gene. This transcriptional cassette was integrated into the chromosomal HIS3 locus. Titrations with 3-AT (3-amino-1,2,4-triazole) indicated that complete a cessation of growth of the host strain harboring the integration occurred with a 15 mM concentration of the inhibitor. Selection assays with a cDNA-activation domain fusion library from [cir⁺] yeast were done at an inhibitor concentration of 45 mM. The positive candidates were subjected to two additional rounds of 3-AT selection before they were subcloned into E. coli and characterized.

Western blot analysis of yeast extracts. Next, 5-ml yeast cultures (optical density at 600 nm of ca. 0.4 to 0.6) were centrifuged, and the pelleted cells were washed twice at 4°C in 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA (pH 8.0), 1 mM phenylmethylsulfonyl fluoride (added from a 200× stock, freshly prepared in isopropanol), and 1× protease inhibitor cocktail from Boehringer Mannheim. The cells were resuspended in 250 µl of the washing buffer and then disrupted by vortexing (six 30-s cycles, with a 30-s intermission on ice between cycles) with acid-washed glass beads (425 to 600 µm in diameter). The supernatants were collected by centrifugation at 4°C in an Eppendorf centrifuge.

In vitro DNA binding assays. A 65-bp synthetic DNA, representing one STB repeat element, was obtained by hybridization of two complementary deoxyoligonucleotides and used as the substrate for the in vitro binding reactions. The Shf1 protein tagged with His-6 was expressed in E. coli from the pTrcHis vector (Invitrogen) and was purified by nickel-affinity chromatography. The protein used in the assays was approximately 50% pure. ³²P-end-labeled DNA (ca. 0.02 pmol) was incubated with roughly 0.4 pmol of the Shf1 protein in 50 mM HEPES (pH 7.5), 50 mM KCl, 5 mM EDTA, and 10% glycerol (14) in the presence or absence of competitor DNA at 30°C for 30 min. Binding reactions were analyzed by electrophoresis in 12% polyacrylamide gels (acrylamide to bisacrylamide, 29:1) under nondenaturing conditions. The DNA-protein complexes were visualized by autoradiography or phosphorimaging.

	RESULTS

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Cellular localization and functional competence of deletion derivatives of Rep1 and Rep2 proteins. Given that the 2µm plasmid exists primarily in the yeast nucleus (with perhaps only occasional excursions outside of it), it is reasonable to suspect that the logical site of action of the proposed Rep1p-Rep2p complex would be the nucleus. Our recent results that the green fluorescence form of GFP-Rep fusions was primarily localized in the nucleus is consistent with this hypothesis (1). The pattern of fluorescence, though, was distinct for Rep1p and Rep2p. Whereas the GFP-Rep1p fluorescence spilled over weakly into the cytoplasm (Fig. 1A), the GFP-Rep2p fluorescence was much more localized and was confined virtually exclusively to the nucleus in the majority of the cells (Fig. 1D). Relatively large amino-terminal deletions of 125 amino acid residues in Rep1p or 150 amino acids in Rep2p did not affect the compartmentation of the corresponding GFP fusions (Fig. 1B and E, respectively). In sharp contrast, very short carboxy-terminal deletions of 25 amino acids in Rep1p (Fig. 1C) or 20 amino acids in Rep2p (Fig. 1F) abolished the nuclear association of fluorescence from these GFP fusion derivatives. The patterns shown in Fig. 1C and F were indistinguishable from the fluorescence pattern seen with GFP alone. The results obtained with all of the Rep deletions tested are summarized in Table 1.

View larger version (23K): [in this window] [in a new window]   FIG. 1.   Fluorescence localization from GFP-Rep1p and GFP-Rep2p in [cir+] and [cir0] cells. The cells were prepared for microscopy after 4 h of induction of the GFP-Rep hybrid proteins by transfer to galactose medium as described by Ahn et al. (1). For each pair of panels A through F, the green fluorescence from GFP-Rep proteins is displayed on the left; the blue nuclear fluorescence from the DAPI-DNA is displayed on the right. Panels A and D represent full-length Rep1p and Rep2p, respectively. Panels B and E represent 125- and 150-amino-acid deletions from the amino termini of Rep1p and Rep2p, respectively. These proteins are referred to here as Rep1pN125 and Rep2pN150. Panels C and F represent deletions of 25 and 20 amino acids from the carboxy termini of Rep1p and Rep2p, respectively (Rep1pC20 and Rep2pC25). The results presented here are for the longest amino-terminal and shortest carboxy-terminal deletions of each of the two Rep proteins. The fluorescence profiles for the other deletions tested are summarized in Table 1.

Restoration of localization and of function in carboxy-terminal Rep deletions by the addition of an NLS sequence. We tested whether the carboxy-terminal deletions of Rep1p and Rep2p can be targeted to the nucleus by the addition of a synthetic heptapeptide derived from the SV40 T antigen that is known to act as an NLS in yeast cells. The results shown in Fig. 2 demonstrate that the two shortest deletions (Rep1C25 and Rep2C20), when provided with the NLS at their carboxy terminus, were efficiently transported to the nucleus. Identical results were obtained when the NLS was placed at the amino terminus of each of these two proteins (data not shown). Furthermore, once they were escorted to their normal cellular locale, both Rep1C25-NLS and Rep2C20-NLS were able to elevate the stability of pSTB2 and pSTB1, respectively, in a [cir⁰] background (Table 3).

View larger version (29K): [in this window] [in a new window]   FIG. 2.   Localization of short carboxy-terminal deletions of Rep1p and Rep2p when provided with the NLS from SV40 T antigen. The fluorescence patterns of Rep1pC25 and Rep2pC20 are shown in panels A and C, respectively, for reference. Panels B and D illustrate the localization of the same deletions when a heptapeptide NLS from an SV40 T antigen was fused to their carboxy termini (B, Rep1pC25-NLS; D, Rep1pC20-NLS). When the NLS was fused to the amino termini of these deletions, the fluorescence localization was indistinguishable from that shown in panels B and D (data not shown).

Interactions among Rep1p and Rep2p and their deletion derivatives assayed in vivo in yeast cells. By using the two-hybrid transcriptional activation assay (8), we tested the interactions among pairwise combinations of full-length Rep proteins and a subset of their deletion derivatives. The "bait" protein, a hybrid between LexA and Rep (or a Rep protein containing a deletion), was expressed constitutively from the yeast ADH promoter harbored by an HIS3 plasmid. The prey, a fusion between Rep (or a Rep protein containing a deletion) and a transcriptional activation domain, was expressed from the inducible GAL1 promoter contained within a TRP1 plasmid. The reporter cassette was a chromosomally integrated copy of the yeast LEU2 gene placed under the control of the LexA operator DNA serving as the UAS (upstream activating sequence). The simultaneous presence of the plasmids expressing the bait and the prey was verified for all of the pairwise combinations, as indicated by the growth of the tester strain in SD medium lacking histidine and tryptophan (Fig. 3, row A). The labels above each column in Fig. 3 represent the two proteins under scrutiny (indicated as the bait [LexA fusion] "+" the prey [the activation domain fusion] above each column). A growing patch in row C (LEU2 reporter; SD medium lacking histidine, tryptophan, and leucine-galactose) indicated a positive interaction between a pair of proteins being tested. Note that, whereas the LexA hybrid would be constitutively expressed (from the ADH promoter), the fusion protein containing the transcriptional activator would be induced only in the presence of galactose (from the GAL1 promoter). As glucose would repress expression of the latter, row B should not support growth. Thus, growth in row C and no growth in row B provided the critical criterion for interaction. The positive control used here, a direct fusion of the activation domain to LexA (i.e., column 1), was an exception to this rule. Since this protein was constitutively made from the ADH promoter, the growth of the host strain in rows C and B (column 1) was expected.

View larger version (42K): [in this window] [in a new window]   FIG. 3.   In vivo baiting assays with Rep1p and Rep2p or their GFP fusion derivatives. Abbreviations: R1, Rep1p; R2, Rep2p; VB, vector containing LexA without Rep fusion; VA, vector containing the transcriptional activation domain without Rep fusion; VP, positive control vector in which the constitutive ADH promoter controlled the expression of the LexA-GAL4 fusion; VN, negative control vector in which LexA fused to a transcriptionally inert protein, Drosophila bicoid, was expressed from the GAL1 promoter. For a given binary protein combination, the protein listed before the plus sign was the bait (fused to LexA), and the protein following the plus sign was the suspected prey (fused to the activation domain). The -galactosidase units shown in the lower panel were obtained in a variation of the dihybrid assay by using lacZ as the reporter gene. The Western blots (lower right panel) were probed with a monoclonal LexA antibody for the bait proteins and with a polyclonal antiserum to the HA1 epitope for the prey proteins.

Interactions of the Rep protein deletions assayed in vitro. The results from the in vivo assays in yeast cells were verified by a GST-Rep hybrid baiting assay with proteins expressed in E. coli (1). In this analysis, GST-Rep1p or GST-Rep2p was used to trap S-tagged Rep1p, Rep2p, or their deletions from supernatants of the bacterial cell extracts containing them. The results of these baiting assays are displayed in Fig. 4. The electrophoretically fractionated samples were probed with S protein to reveal Rep1p, Rep2p, or their deletions harboring this tag. The presence of each of the S-tagged proteins in the appropriate E. coli extracts used for baiting is shown for reference (Fig. 4A and B, lanes 2, 3, and 4; Fig. 4C, lane 2). When GST-Rep1p was used as the bait, S-Rep1p or S-Rep2p could be pulled down from the respective supernatants (Fig. 4A and B, lanes 5). Similar results were obtained in the reciprocal experiment with GST-Rep2p as the bait (Fig. 4A and B, lanes 8). These results are in conformity with the previous observations of Ahn et al. (1). In the case of the S-tagged deletion proteins, positive interaction was observed between GST-Rep2p and Rep1pC140 (Fig. 4A, lane 10). Furthermore, the S-tagged amino-terminal 150-amino-acid peptide from Rep1p (S-Rep1pC223) was able to interact with GST-Rep2p (Fig. 4C, lane 4), but not with GST-Rep1p (lane 3). Control assays with GST alone failed to trap any of the S proteins tested (Fig. 4A and B, lanes 11 to 13; Fig. 4C, lane 5).

View larger version (35K): [in this window] [in a new window]   FIG. 4.   Baiting assays for Rep1p and Rep2p and their deletions performed with GST-Rep1p and GST-Rep2p hybrid proteins. The assays were carried out as described by Ahn et al. (1). After baiting, proteins were fractionated in SDS-12% polyacrylamide gels and probed with the S-protein probe. The lane marked M displays the molecular-weight standards. (A and B) In lanes 2 to 4, proteins extracted by boiling the cells from 200-µl portions of the induced cultures (that were also the starting materials for the assays depicted in lanes 5 to 13) in the SDS sample buffer were run as controls. The S-Rep protein bands of interest in lanes 2 to 4 are indicated by the arrowheads. The data in panel A were obtained with S-tagged Rep1p or its derivatives; the data in panel B were obtained with S-tagged Rep2p or its derivatives. The GST-Rep hybrids used as the baits and the corresponding baited S-Rep proteins (either full length or partially deleted) are indicated above lanes 5 to 10. In the reactions represented in lanes 11 to 13, the baiting was performed with GST and not with the GST-Rep hybrids. (C) Results obtained with Rep1pC223 fused to the S-peptide tag. Lane 2 represents the extract from 200 µl of the induced culture expressing Rep1pC223; lanes 3 to 5 correspond to baiting assays with GST-Rep1p, GST-Rep2p, and GST, respectively. Detailed protocols of the assays are described in Ahn et al. (1).

Rep1p, Rep2p, and a host-encoded protein interact with the STB locus in vivo. It has been hypothesized that the STB locus must directly or indirectly interact with the Rep proteins in mediating plasmid stability. The evidence in support of this notion is circumstantial at best. It is clear that the trans-acting Rep proteins and the cis-acting STB locus are functionally tightly interrelated, since mutations in any one of the three loci (REP1, REP2, or STB) lead to the same phenotype, namely, plasmid instability. Preliminary in vitro binding studies (14) have suggested that Rep1p and Rep2p can bind to STB but only in association with a host factor (or factors).

View larger version (90K): [in this window] [in a new window]   FIG. 5.   Chromosomally encoded yeast protein Shf1p that interacts with the STB locus. The monohybrid assay identified the STB-interacting host protein as the product of the YIL036W locus (from the yeast genome bank; now designated as SHF1). The complete nucleotide sequence of the genetically and biochemically uncharacterized SHF1 gene, along with the amino acid sequence of the presumed protein product encoded by it, are shown. The rectangular box (from Phe-377 to Ser-294) indicates a potential GTP or ATP binding sequence (prediction by the UCLA-DOE structure prediction server). The shaded box (from Trp-421 to Phe-454) is highly homologous to the consensus CREB motif present in the ATF/bZIP family of transcriptional regulatory proteins. (For a comparison of this region to homologous regions in a selected set of eukaryotic CREB/bZIP proteins, see Fig. 6.) The abundance of glutamine residues in the hatched box (Gln-118 to Gln-135) is suggestive of the activation patches in a number of transcription factors (6, 7).

View larger version (43K): [in this window] [in a new window]   FIG. 6.   The CREB motif in the STB-interacting yeast protein Shf1p shows strong homology to the basic and zipper regions of transcriptional regulators from eukaryotes. In the sequence alignment of the Trp-421 to Phe-454 region of Shf1p with similar regions from other eukaryotic ATF/bZIP proteins, amino acid identities or strong conservations are indicated in boldface. The GCN4 and ACR1 proteins are from S. cerevisiae. The MTS proteins are from Schizosaccharomyces pombe, and the ATF series of proteins, CREM and TGA1, are from other organisms. The numbers in parentheses indicate the relevant references.

The Shf1 protein binds to the STB locus in vitro. The results from in vitro assays for the binding of Shf1p to the STB locus are shown in Fig. 7. In the 2µm plasmid, the STB sequence is present as a tandem, directly oriented array of 5 to 6 U of a 65-bp consensus element. The binding reactions were done by using a single repeat unit as the substrate. Association between Shf1p and the 65-bp STB element yielded a series of DNA-protein complexes (Fig. 7, lanes 2). This finding suggests that Shf1p may bind to DNA as oligomeric units or that, alternatively, the Shf1p-bound STB elements may associate with each other to produce the hierarchical binding pattern. No specific complexes were observed when binding was done with crude extracts (or extracts fractionated over the nickel column) from IPTG (isopropyl--D-thiogalactopyranoside)-induced E. coli cells harboring the expression vector without the SHF1 clone (data not shown). The specificity of Shf1p-STB binding was tested by challenge with non-STB DNA: a 60-bp deoxyoligonucleotide harboring the target site for the 2µm plasmid encoded Flp recombinase (Fig. 7A, lanes 3 to 7) or salmon sperm DNA (Fig. 7B, lanes 3 to 7). At a ca. 5 or 10 M excess of the Flp binding sequence over STB (0.02 pmol per reaction), little or no competition was observed (Fig. 7A, lanes 3 and 4). As the concentration of the competitor was increased, the yield of the higher-order complexes diminished (lanes 5 to 7). However, even at a competitor/STB molar ratio of 250:1, binding was not abolished (lane 7). Comparable results were obtained when salmon sperm DNA was used as the competitor (Fig. 7B). The pattern of competition obtained with 0.15 µg of salmon sperm DNA (equivalent to ca. 5 to 6 pmol of a 60-bp-long DNA) was roughly equivalent to that seen with similar amounts of the Flp DNA target (Fig. 7B, compare lanes 3 and 7). Binding was still evident when the molar ratio of salmon sperm DNA to STB was raised to approximately 4,000:1 (lane 7).

View larger version (59K): [in this window] [in a new window]   FIG. 7.   Binding of Shf1p in vitro to the STB DNA elements. Approximately 0.02 pmol of the STB DNA (32P-labeled at the 5' end) was incubated with Shf1p (ca. 0.4 pmol) as described in Materials and Methods. Binding reactions were fractionated by electrophoresis in 12% nondenaturing polyacrylamide gels. For both panels, reactions in lane 1 were controls without the addition of Shf1p; those in lane 2 did not contain competitor DNA. Reactions in lanes 3 to 7 contained the indicated amounts of the competitor DNA: a 60-bp synthetic oligonucleotide in panel A and salmon sperm DNA in panel B. The unbound substrate is marked (S). The bound complexes are denoted by S-Shf1p.

Plasmid stability in a host strain harboring a deletion of the SHF1 locus. Does the lack of SHF1 function affect the stability of a 2µm circle-derived test plasmid? To address this issue, the maintenance of plasmid pSTB (harboring the 2µm plasmid replication origin and the STB locus) was followed in a [cir⁺] SHF1 strain (Fig. 8A) and a derivative strain lacking this locus (Fig. 8B) (see Materials and Methods for details). The plasmid loss rate was significantly higher in the shf1 deletion strain, as indicated by the increase in the number of red colonies (indicating the loss of the plasmid-borne ADE2 marker). The results are also expressed as the relative SI (Fig. 8). When the red colonies were transferred to SD plates lacking leucine, they did not grow, thus verifying the simultaneous loss of the ADE2 and LEU2 markers carried on the plasmid pSTB.

View larger version (118K): [in this window] [in a new window]   FIG. 8.   Maintenance of a 2µm circle-derived plasmid in a host strain deleted for the SHF1 locus. (Top) The stability of the test plasmid pSTB (harboring the ADE2 and LEU2 markers) was assayed as described in Materials and Methods. The host strains (wild type for SHF1 in panel A and shf1 in panel B) containing pSTB were grown nonselectively for approximately 10 generations and plated on YEPD plates to assay plasmid loss by colony color. (Bottom) The results from four separate transformants are expressed as the SI as follows: SI = (the number of white colonies/total number of red and white colonies) × 100. A sectored colony was scored as red if the red sector was one-fourth the colony size or larger; otherwise, it was counted as white.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

The 2µm circle plasmid in yeast cells employs a dual strategy for its stable propagation as a benign parasite genome: efficient plasmid partitioning and copy number amplification. Genetic experiments (16, 19, 24, 25, 28) have given rise to a model in which the components of the stability system provide a measure of the plasmid copy number and contribute to the transcriptional regulation of the amplification system (see Fig. 9). Previous experiments (1) have substantiated one central aspect of the model: the assumption that REP1 and REP2 gene products, the two plasmid-coded trans-acting components of the stability system, must physically interact. The analyses of Ahn et al. revealed that the two proteins not only interact in vivo in yeast cells and are targeted to the nucleus but also associate with each other in vitro in the absence of any other yeast proteins. We have now localized the nuclear targeting sequences within Rep1p and Rep2p, functionally replaced them with an exogenous NLS, and mapped (by in vivo and in vitro assays) the peptide regions responsible for Rep protein associations. Furthermore, our studies provide a molecular framework for examining the role of the cis-acting STB locus and of host factors in conferring plasmid stability.

View larger version (15K): [in this window] [in a new window]   FIG. 9.   Model for protein-protein and DNA-protein interactions in the high-copy maintenance of the 2µm plasmid. The plasmid is shown in its standard representation, with the parallel lines indicating the 599-bp inverted repeats. The results of early analyses, primarily genetic assays, suggested a potential bipartite Rep1p-Rep2p (R1-R2) regulator that represses transcription from the REP1, FLP, and RAF1 promoters. Direct evidence for the interaction between Rep1p (R1) and Rep2p (R2) in vivo and in vitro was obtained recently (1). The notion that interaction between the cis-acting STB locus and the Rep proteins may be required to promote plasmid partitioning at cell division has been entertained and has received limited experimental support (14). The present study presents evidence for the binding of Rep1p, Rep2p, and a host protein Shf1p to the STB locus. There is some indication that product of the RAF1 gene (D) may antagonize the Rep1p-Rep2p-mediated transcriptional repression. This may be a primary step in triggering copy number amplification of the plasmid. The product of the FLP gene (F) is a site-specific recombinase that is essential for the amplification mechanism (11, 33). The plasmid replication origin is denoted by ORI. The 1,950-nucleotide 2µm circle transcript that originates at STB and spans the REP1 locus is indicated. The diagram, adapted from Ahn et al. (1), summarizes contributions from several laboratories and includes the results of this study (1, 3, 9, 14, 16, 19, 24, 25, 28, 33).

The carboxy-terminal regions of the Rep proteins are essential for their localization. The cytological assays with GFP and hybrid proteins have revealed that the nuclear compartmentation signals in the Rep proteins are located at the very carboxy-terminal ends of these proteins. Deletions as short as 25 and 20 amino acids from the carboxy-terminal end (of Rep1p and Rep2p, respectively) are sufficient to delocalize them in the cell and to render them nonfunctional in plasmid maintenance. Their capacity for nuclear homing and their activity in plasmid maintenance can be fully restored by an NLS sequence derived from the SV40 T antigen placed either at the amino terminus or at the carboxy terminus. There is some qualitative difference between the patterns of nuclear localization of native Rep1p and the deletion Rep1p harboring the SV40 NLS (compare Fig. 2B to Fig. 1A). This difference, though, is apparently irrelevant to the role of Rep1p in plasmid stability.

Mapping of the Rep1p-Rep2p interaction domains. The in vivo dihybrid assays and the in vitro GST fusion baiting assays described here have shown the amino-terminal portion of Rep1p consisting of 150 amino acids to be sufficient for interacting with Rep2p. Among the Rep protein deletions in our collection, we have not seen any that have retained the ability for self-interaction. Nor have we identified a deletion of Rep2p that is capable of interacting with the native Rep1p or its deletion variants. It should be clarified that the lack of association between a carboxy-terminal deletion variant of Rep with its full-length version or with the partner Rep (Rep2p in the case of a Rep1p deletion and Rep1p in the case of a Rep2p deletion) cannot be due solely to a lack of the NLS resulting from the deletion. Note that the expression plasmids in the dihybrid system (8) have built-in NLS sequences that are fused in frame to the coding sequences of the proteins being analyzed.

Potential role of host factors in the stable maintenance of the 2µm plasmid. The isolation of a chromosomally encoded yeast protein Shf1p in an assay designed to select for proteins that bind to the STB locus suggests the potential involvement of a host factor (or host factors) in the persistence of the 2µm plasmid as a benign parasite genome in yeast cells. Note that the same assay has also identified Rep1p and Rep2p as the other two yeast proteins that interact with STB. The Shf1 protein, expressed in E. coli, binds to the STB locus with high specificity, and a host strain lacking SHF1 is compromised in its capacity to maintain a 2µm circle-derived plasmid. The Shf1 protein could well recruit the Rep proteins to the STB locus. Such an activity would agree with the in vitro results (14) that suggest the involvement of a host factor in the binding of the Rep proteins to STB. Whether there is a direct interaction between the Shf1 protein (selected in the monohybrid-3-AT assay) and one or both of the Rep proteins is not known at present.

Summary. As has been pointed out previously, the circular geometry, structural compactness, and functional parsimony of the 2µm plasmid appear to represent an optimized evolutionary solution for the high-copy maintenance of an extrachromosomal selfish DNA element in yeast cells (2). The general picture of the suspected protein-protein and protein-DNA interactions that contribute to this phenomenon, at least some of which have received experimental support, is given in Fig. 9. In a recent study (1) we established by a variety of independent assays that Rep1p and Rep2p can directly interact with each other. We have now extended the potential significance of this finding by demonstrating the interaction of the STB locus with Rep1p, Rep2p, and the product of the chromosomal gene SHF1. The results of the present study therefore offer new insight into the analysis of the molecular contributions made by the host and the plasmid to this benign parasitism.