The yeast 2-μm plasmid Raf protein contributes to plasmid inheritance by stabilizing the Rep1 and Rep2 partitioning proteins

Abstract

The yeast 2-μm plasmid is a remarkable genetic parasite, managing efficient maintenance at high-copy number with minimal impact on the host. Equal partitioning of the plasmid upon host cell division requires plasmid proteins Rep1 and Rep2 and the plasmid STB locus. The Rep proteins and the plasmid-encoded Raf protein also regulate plasmid gene transcription. In this study, protein interaction assays, sequence analyses and mutational approaches were used to identify domains and residues in Rep2 and Raf required for association with Rep1 and Rep2 and to delineate the Rep2 DNA-binding domain. Rep2 and Raf displayed similarities in interactions with Rep1 and Rep2, in having Rep1 promote their STB association in vivo, and in stabilizing Rep protein levels. Rep2 mutants impaired for self-association were competent for transcriptional repression while those deficient for Rep1 association were not. Surprisingly, Rep2 mutants impaired for either Rep1 interaction or self-association were able to maintain efficient plasmid inheritance provided Raf was present and competent for Rep protein interaction. Our findings provide insight into the Rep protein complexes required for partitioning and transcriptional repression, and suggest that in addition to its transcriptional function, Raf stabilization of Rep partitioning proteins contributes to the remarkable persistence of the 2-μm plasmid.

Figure 1.

Model for interaction of 2-μm plasmid proteins with plasmid loci. The B form of the 2-μm plasmid is shown with positions of plasmid genes (white arrows), inverted repeat sequences (IR; gray boxes with arrows showing orientation), origin of replication (ARS; black box) and STB (striped box) indicated. Interactions of Rep1, Rep2 and Raf proteins with the 2-μm plasmid are shown. Rep1 and Rep2 associate with STB to mediate plasmid partitioning (9,11,16), and repress transcription driven by STB and divergent 2-μm plasmid promoters (23,25,26), with Raf acting to relieve this repression (26).

Figure 2.

Raf associates with Rep1 and Rep2 independently and displays overlap with Rep2 in the regions required for these associations. (A–C) A cir⁰ two-hybrid reporter strain was co-transformed with two plasmids, one encoding Gal4_AD (AD), or Gal4_AD fused to Rep1, Rep2 or Raf; and the other encoding LexA_BD (BD), or LexA_BD fused to full-length or truncated versions of Raf (A), Rep1 (B) or Rep2 (C). Interaction of the two fusion proteins, indicated by activation of expression of the lacZ reporter gene, was monitored using a filter assay with the substrate X-gal, which yields a dark-coloured precipitate when cleaved by ß-galactosidase. (D) Hexahistidine-tagged thioredoxin (Trx) and Trx-Raf, Trx-Rep2_199–296 and Trx-Rep2_232–296 were purified from Escherichia coli cell lysates by metal ion affinity chromatography and resolved in duplicate gels by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Proteins were visualized by staining with Coomassie Blue (top) or transferred to a nitrocellulose membrane followed by incubation with a ³²P-radiolabeled STB 63-bp repeat probe. DNA binding was detected by autoradiography (bottom). (E) The regions of Rep1, Rep2 and Raf involved in each of the indicated interactions and for Rep2 with DNA are summarized. Residues shown for Rep1 self-association are based on prior in vitro studies (15) and two-hybrid interaction assays (Supplementary Figure S2).

Figure 3.

Identification of amino acid substitutions in Rep2 and Raf that selectively impair interaction with Rep1 or Rep2. A cir⁰ two-hybrid reporter strain was co-transformed with two plasmids, one expressing Gal4_AD (AD) or Gal4_AD fused to Rep1, Rep2 or Raf and the other expressing LexA_BD (BD) or LexA_BD fused to wild-type or mutant Rep2 (A and C) or Raf (B and D). (A and B) The two-hybrid assay for protein–protein interaction was performed as described in the legend for Figure 2. (C and D) Total protein was extracted from the co-transformants shown in (A) and (B) respectively, and equal amounts from each analyzed by western blotting. BD-fusions were detected with anti-LexA with the exception of BD-Rep2 expressed with AD-Raf where they were detected with anti-Rep2. Blots were stripped and reprobed for AD fusions with anti-Gal_AD antibody with the exception of AD-Rep1 fusions where duplicate blots were probed with anti-Rep1. Asterisks denote non-target host proteins detected by the antibodies.

Figure 4.

Rep2 transcriptional repressor activity is retained by Rep2_AA but not by Rep2_D22N, with Raf_F18S but not Raf_AA able to alleviate this repression. The indicated Rep1, Rep2 and Raf proteins were expressed under the control of galactose-inducible promoters from ARS/CEN pRSGAL-based plasmids in a cir⁰ strain containing a FLP promoter-lacZ fusion reporter gene (FLPp-lacZ) integrated in the genome. (A) Expression of the FLPp-lacZ reporter was monitored by assaying for ß-galactosidase activity after 24 h of growth in selective medium containing galactose. Results represent the average (±s.d.) from analyzing four independent transformants. (B) Total protein extracted from the strains in (A) expressing Rep1, and either no Rep2 or the indicated form of Rep2, was analyzed by western blotting with antibodies specific for Rep1, Rep2 and Pgk1. Rep2-P is a hyperphosphorylated form of Rep2.

Figure 5.

Raf is required for partitioning competence of Rep2_D22N and Rep2_AA. A cir⁰ yeast strain was transformed with kanMX4-tagged, amplification-incompetent (flp⁻) 2-μm plasmids (pKan-based) carrying a wild-type REP1 gene and the indicated version of the REP2 and RAF genes. Due to the absence of a functional FLP gene, plasmid missegregation events cannot be corrected by Flp-mediated copy number amplification, making efficient maintenance of the pKan-based plasmids dependent on Rep protein partitioning function. Transformants were cultured overnight (six to eight generations) in selective medium, and the fraction of plasmid-bearing cells determined by a plating assay. Results represent the average (±s.d.) of five independent transformants for each plasmid. Plasmid copy number in each culture was determined by polymerase chain reaction (PCR) using total DNA extracted from the transformants as template and quantifying the ratio of product obtained with primers specific for a plasmid relative to a chromosomal locus. This value was then corrected for the fraction of cells in the population containing plasmid to obtain the average plasmid copy number per plasmid-bearing cell (See Supplementary Figure S6 for details).

Figure 6.

Raf increases Rep1 and Rep2 protein levels. A cir⁰ yeast strain was transformed with two ARS/CEN galactose-inducible expression plasmids: one (pBM272-based) expressing either no protein, or expressing Rep1 and Rep2 variants individually or simultaneously; and the other expressing no protein (pRSGAL-LEU), or expressing Raf (pRSGAL-LEU-RAF). Transformants were cultured overnight in selective medium containing galactose to induce protein expression. Total protein was analyzed by western blotting with antibodies specific to Rep1, Rep2 and Pgk1. Rep2-P is a hyperphosphorylated form of Rep2. For each cell extract, presence (+) or absence (−) of the wild-type version of each protein, or presence of a mutant version of Rep2 (D22N or AA) is indicated above the lane.

Figure 7.

Raf associates with STB and 2-μm plasmid gene promoters, and association with STB is dependent on Rep1. (A) A cir⁺ yeast strain was transformed with an ARS/CEN plasmid expressing FLAG-tagged Raf under the control of a galactose-inducible promoter. Transformed yeast were cultured in medium containing galactose and chromatin was immunoprecipitated with antibodies specific for native Rep1 and Rep2, anti-FLAG (FLAG-Raf) and, as a negative control, anti-HA. The precipitated DNA was analyzed by semi-quantitative PCR with primers specific for the STB locus, for the divergent FLP/REP2 and REP1/RAF promoter regions (FLP/REP2p and REP1/RAFp), and, as a negative control, a chromosomal locus (TRP1). The bar graph indicates ChIP efficiency as the percent of input DNA immunoprecipitated; results are average (±s.d.) from triplicate assays. Ethidium bromide-stained agarose gels of PCR products from a representative assay are shown below the graph. Template DNA amplified in ‘input’ PCR reactions is 10% of that amplified in ‘ChIP’ PCR reactions. Products obtained from neat and 1:4 dilutions of each template are shown. (B) A cir⁰ yeast strain with STB integrated in the chromosome upstream of a HIS3 reporter gene was co-transformed with two galactose-inducible expression plasmids: one expressing B42_AD-HA (AD) or B42_AD-HA-Raf (AD-Raf), and the second, expressing either no protein (−), or expressing Rep1 or Rep2. Five-fold serial dilutions of co-transformants were spotted onto solid media that selected for the presence of the two plasmids, with galactose to induce Rep protein expression, and either containing histidine (+His), or lacking histidine (−His) and containing 3-aminotriazole (3-AT). Growth on the −His + 3-AT medium indicates recruitment of the AD fusion protein to STB.

Figure 8.

Overexpression of RAF impairs Rep2 association with STB. A cir⁺ yeast strain with STB integrated in the genome upstream of a HIS3 reporter gene was co-transformed with two galactose-inducible expression plasmids: one that expressed either B42_AD-HA (AD), or Rep1 or Rep2 fused to B42_AD-HA (AD-Rep1 or AD-Rep2); and the second expressing no protein (−) or expressing Raf. Recruitment of AD fusion proteins to STB was monitored as described in the legend of Figure 7.

Figure 9.

Models for the effect of Raf and loss of specific Rep2 associations on Rep protein complexes at the 2-μm plasmid FLP gene promoter and STB partitioning locus. (A) In the absence of Raf (top), FLP gene transcription is repressed by co-expression of wild-type Rep1 with wild-type Rep2 or with Rep2_AA, suggesting that a Rep1–Rep2 dimer associating with the FLP promoter is sufficient for repression. Expression of Rep1 in the absence of Rep2, or with Rep2_D22N does not repress FLP transcription, consistent with a complex of Rep1 with Rep2 being required for repression, although low Rep1 protein levels in the absence of Rep2 association preclude determining whether Rep1 alone would be sufficient to block transcription. The presence of Raf (bottom) relieves Rep protein-mediated FLP repression, likely by competing with Rep2 for association with Rep1, and stabilizes Rep protein levels. (B) Wild-type Rep1 and Rep2 proteins, in association with the STB locus, are sufficient to mediate plasmid partitioning in both the absence (top) and presence (bottom) of Raf. Interaction is shown between the Rep2 subunits of the Rep1–Rep2 heterodimer at left but may not be essential for establishing the functional partitioning complex at STB, as indicated by Rep2 mutants lacking this interaction. When Rep2 association with Rep1 or Rep2 is impaired (as is the case for the Rep2_D22N and Rep2_AA mutants, respectively), partitioning function is impaired unless Raf is present. Raf may compensate for these impaired associations by stabilizing Rep protein levels. Raf might also contribute more directly to the partitioning complex by replacing Rep2 at a subset of sites in the repeated STB sequence; however, Raf is not sufficient to provide partitioning function in the absence of Rep2, despite stabilizing Rep1 protein levels. The potential role of Rep1 self-association in Rep-mediated transcriptional repression and partitioning is not shown in these models and remains to be determined. Proteins present at levels lower than those observed when Rep2 is wild-type are shown with a dashed line with more significantly reduced levels indicated by lighter shading.