The 2 micron plasmid purloins the yeast cohesin complex: a mechanism for coupling plasmid partitioning and chromosome segregation?

Abstract

The yeast 2 micron plasmid achieves high fidelity segregation by coupling its partitioning pathway to that of the chromosomes. Mutations affecting distinct steps of chromosome segregation cause the plasmid to missegregate in tandem with the chromosomes. In the absence of the plasmid stability system, consisting of the Rep1 and Rep2 proteins and the STB DNA, plasmid and chromosome segregations are uncoupled. The Rep proteins, acting in concert, recruit the yeast cohesin complex to the STB locus. The periodicity of cohesin association and dissociation is nearly identical for the plasmid and the chromosomes. The timely disassembly of cohesin is a prerequisite for plasmid segregation. Cohesin-mediated pairing and unpairing likely provides a counting mechanism for evenly partitioning plasmids either in association with or independently of the chromosomes.

Figure 1.

Partitioning of STB plasmids and ARS plasmids in yeast mutants that are defective in chromosome segregation. The construction of STB and ARS plasmids and their functional differences have been detailed earlier (Velmurugan et al., 2000). They are virtually indistinguishable in their segregation properties in a [cir⁰] host strain (lacking the Rep proteins). However, in a [cir⁺] strain (providing Rep proteins in trans), they behave quite distinctly. (A) The temperature-arrested cells with missegregated chromosomes were categorized into two types: a and b. The representative cells shown here are from the ctf13-30 strain. The chromosome and plasmid profiles were scored by DAPI and green fluorescence, respectively. The values for each cell type were derived from ∼450 largely budded cells for each strain. (B) The nuclear membrane and the mitotic spindle were visualized by fluorescence microscopy in live yeast cells expressing Nup49p–CFP and Tub1p–YFP simultaneously. Red and green colors (tubulin and nuclear membrane, respectively) were added artificially using the Adobe Photoshop^® software. The patterns shown here are representative of ∼80% of the cells shifted to the restrictive temperature.

Figure 7.

Distinction between the 2 micron plasmid and the chromosomes in binding G1-expressed Mcd1p. Myc-tagged Mcd1p was overexpressed from the GAL10 promoter in G1-arrested cells. (A) Chromosome spreads were probed for Rep1p, Mcd1p, or the STB-containing plasmid pSV1 by immunostaining. Antibodies to native Rep1p, the Myc epitope (fused to Mcd1p), or the Lac repressor bound to the operator repeats on pSV1 were used. The signal from Mcd1p expressed inappropriately in G1 was weak. Images obtained with an Optronix Quantix camera were deconvolved and enhanced using the Metamorph software. (B and C) The same antibodies to Mcd1p as in A were used in the chromosome immunoprecipitation assays. Primers specific to STB, a cohesin site on chromosome V, or CEN3 were used in PCR amplifications.

Figure 7.

Distinction between the 2 micron plasmid and the chromosomes in binding G1-expressed Mcd1p. Myc-tagged Mcd1p was overexpressed from the GAL10 promoter in G1-arrested cells. (A) Chromosome spreads were probed for Rep1p, Mcd1p, or the STB-containing plasmid pSV1 by immunostaining. Antibodies to native Rep1p, the Myc epitope (fused to Mcd1p), or the Lac repressor bound to the operator repeats on pSV1 were used. The signal from Mcd1p expressed inappropriately in G1 was weak. Images obtained with an Optronix Quantix camera were deconvolved and enhanced using the Metamorph software. (B and C) The same antibodies to Mcd1p as in A were used in the chromosome immunoprecipitation assays. Primers specific to STB, a cohesin site on chromosome V, or CEN3 were used in PCR amplifications.

Figure 7.

Distinction between the 2 micron plasmid and the chromosomes in binding G1-expressed Mcd1p. Myc-tagged Mcd1p was overexpressed from the GAL10 promoter in G1-arrested cells. (A) Chromosome spreads were probed for Rep1p, Mcd1p, or the STB-containing plasmid pSV1 by immunostaining. Antibodies to native Rep1p, the Myc epitope (fused to Mcd1p), or the Lac repressor bound to the operator repeats on pSV1 were used. The signal from Mcd1p expressed inappropriately in G1 was weak. Images obtained with an Optronix Quantix camera were deconvolved and enhanced using the Metamorph software. (B and C) The same antibodies to Mcd1p as in A were used in the chromosome immunoprecipitation assays. Primers specific to STB, a cohesin site on chromosome V, or CEN3 were used in PCR amplifications.

Figure 2.

Localization of the 2 micron plasmid, the Rep proteins, and the Mcd1 protein in yeast chromosome spreads. Chromosomes were visualized by DAPI, and proteins by immunofluorescence from fluorescein or Texas red–conjugated secondary antibodies. The reporter plasmid (pSV1) harbored Lac operator DNA, and could be revealed by the bound Lac repressor using indirect immunofluorescence. The asterisk over pSV1 (bottom panel of B) denotes that the plasmid fluorescence was artificially changed from green to red using Adobe Photoshop^® software for the purpose of overlaying it on the DAPI fluorescence.

Figure 3.

ChIP assays using antibodies directed to the Mcd1 protein. The assays were performed with monoclonal antibodies to the HA epitope tag fused to Mcd1p. The relevant genetic features of the yeast strains ([cir⁰] or [cir⁺]; presence of ARS- or STB-containing reporter plasmids; expression of Rep proteins) are indicated above the experimental panels. The schematic representation of the 2 micron plasmid genome at the bottom shows the location of the origin (ORI) and the open reading frames (REP1, REP2, FLP, and RAF1) with respect to STB. The inverted repeats (IR1 and IR2) contain the Flp recombination target sites (FRT1 and FRT2). The regions probed by PCR are denoted by the horizontal bars. The experimental lanes are captioned ChIP. WCE refers to whole cell extracts from which DNA was PCR amplified without immunoprecipitation. The mock-immunoprecipitated negative controls are indicated by “Beads.”

Figure 4.

Probing for STB and CEN sequences in chromatin immunoprecipitates obtained with antibodies to Rep1p, Rep2p, Mcd1p, or kinetochore proteins. The proteins targeted for immunoprecipitation are indicated above the respective experimental panels: Rep1p in A, C, and E; Rep2p in B and F; Mcd1p–HA in D; and Ndc10p–HA and Ndc80p–HA in G and H, respectively. As in Fig. 3, the relevant features of the yeast strains are also shown.

Figure 5.

Monohybrid tests for Mcd1p– STB association and ChIP assays for Smc1p–STB and Smc3p–STB association. (A) The monohybrid assays were performed in isogenic [cir⁰] and [cir⁺] strains as described previously (Velmurugan et al., 1998). AD, activation domain. (B and C) ChIPs were done with antibodies to the Myc epitope (B) or the HA epitope (C).

Figure 6.

Cell cycle dependence of Mcd1p binding to STB. Time zero refers to the release of G1-arrested cells from α factor. Representative DIC images of the cells at the different time points after removing the cell cycle arrest are shown at the bottom. The DNA contents of corresponding cell samples derived by FACS^® analysis are shown at the right.

Figure 8.

Effect of noncleavable Mcd1p on the partitioning of an STB plasmid and an ARS plasmid. Small budded cells from an exponentially growing culture, induced for GFP–Lac repressor expression were followed by time-lapse fluorescence microscopy for 90 min in dextrose medium or for 150 min after shifting to galactose medium. The representative fluorescence patterns at the initial and final time points are shown. For each experiment, the number of cells that displayed a given pattern is expressed as a fraction of the total number of cells assayed.

Figure 9.

A model for the role of the cohesin complex in the segregation of the 2 micron plasmid. The clustering of the 2 micron plasmid and the compactness of the cluster are mediated by the plasmid stability system and are not dependent on cohesin. The duplication and partitioning of the plasmid cluster may occur via mechanisms depicted in A or B or variations of these. (A) Cohesin-mediated pairing of replicated plasmid clusters occurs during the S phase as does the pairing of sister chromatids. The plasmid clusters are tethered to sister chromatids, likely by a cohesin- independent mechanism. (B) Cohesin-mediated pairing and unpairing are common to the chromosomes and the plasmid clusters as in A. The plasmids are partitioned, however, without physical attachment to the chromosomes.