In vivo analysis of cohesin architecture using FRET in the budding yeast Saccharomyces cerevisiae

Abstract

Cohesion between sister chromatids in eukaryotes is mediated by the evolutionarily conserved cohesin complex. Cohesin forms a proteinaceous ring, large enough to trap pairs of replicated sister chromatids. The circumference consists of the Smc1 and Smc3 subunits, while Scc1 is thought to close the ring by bridging the Smc (structural maintenance of chromosomes) ATPase head domains. Little is known about two additional subunits, Scc3 and Pds5, and about possible conformational changes of the complex during the cell cycle. We have employed fluorescence resonance energy transfer (FRET) to analyse interactions within the cohesin complex in live budding yeast. These experiments reveal an unexpected geometry of Scc1 at the Smc heads, and suggest that Pds5 plays a role at the Smc hinge on the opposite side of the ring. Key subunit interactions, including close proximity of the two ATPase heads, are constitutive throughout the cell cycle. This depicts cohesin as a stable molecular machine undergoing only transient conformational changes during binding and dissociation from chromosomes. Using FRET, we did not observe interactions between more than one cohesin complex in vivo.

Figure 1

Establishment of FRET to analyse proximity between cohesin subunits in budding yeast. (A) In vivo concentrations of five budding yeast cohesin subunits. Nuclear fluorescence intensities were measured in yeast strains Y3087 (MATa/α SCC1-CFP), Y2490 (MATa/α SCC3-CFP), Y2489 (MATa/α PDS5-CFP), Y1967 (MATa/α SMC1-CFP) and Y1971 (MATa/α SMC3-CFP). Error bars represent s.d. (n⩾50 for each strain). (B) Positive and negative FRET controls. Strains Y2588 (MATa/α SCC3-YFP-CFP) and Y2587 (MATa/α PDS5-YFP-CFP) were subject to FRET analysis. Fluorescence in the YFP, FRET and CFP channels is shown, as well as the FRET_R values derived, as described in Materials and methods. Strains containing fluorophore pairs at the Scc3 and Pds5 C-termini, Y2575 (MATa/α SCC3-YFP PDS5-CFP) and Y2574 (MATa/α SCC3-CFP PDS5-YFP), showed fluorescence intensities in the FRET channel close to what is expected from spectral spillover alone.

Figure 2

Constitutive proximity of the Smc1 and Smc3 ATPase heads. (A) Close proximity of fluorophore pairs attached to the Smc1 and Smc3 heads. FRET was analysed in exponentially growing cells of strains Y1966 (MATa/α SMC1-YFP SMC3-CFP) and Y1972 (MATa/α SMC1-CFP SMC3-YFP). (B) Constitutive Smc1/Smc3 head proximity throughout the cell cycle. Small unbudded G1 cells of strain Y1972 were isolated by centrifugal elutriation and released to progress through a synchronous cell cycle. Samples for FRET analysis were processed every 30 min. Cell cycle progression was monitored by FACS analysis of DNA content. (C) Scc1-independent association of the Smc1/Smc3 heads. Strain Y2864 (MATa/α SMC1-YFP SMC3-CFP GAL1-SCC1-HA3) was grown in galactose containing medium and one-half of the culture as transferred to medium lacking galactose, to repress Scc1 expression. After 2 h Scc1 levels and FRET were analysed. (D) Smc head proximity is maintained after cohesin dissociation from chromosomes. Cells of strain Y3254 (MATa/α SMC1-CFP SMC3-YFP GAL1-SCC1(met269-566)) were grown in YP raffinose medium, and arrested in G2/M by nocodazole treatment. Expression of the Scc1 C-terminal fragment was induced by galactose addition for 2 h, and confirmed by Western blotting (data not shown). FRET was analysed in these and control cells that were left without galactose. Images show redistribution of cohesin from nuclear foci after expression of the Scc1 cleavage fragment.

Figure 3

Scc1 and Scc3 association with the cohesin complex. (A) An unexpected geometry of Scc1 at the Smc heads. FRET measurements in strains Y2480 (MATa/α SCC1-YFP SMC1-CFP), Y2481 (MATa/α SCC1-YFP SMC3-CFP), Y2482 (MATa/α SCC1-CFP SMC3-YFP), Y2483 (MATa/α SCC1-CFP SMC1-YFP), Y2593 (MATa/α CFP-SCC1 SMC1-YFP) and Y2594 (MATa/α CFP-SCC1 SMC3-YFP) show that the Scc1 C-terminus is placed close and equidistant to both Smc1 and Smc3 heads. A cartoon illustrates the geometry derived from these data. (B) Mapping of Scc3 to the cohesin complex. FRET was analysed, from left to right, in strains Y2589, Y2533, Y2721, Y2704, Y2534, Y2591, Y2722 and Y2723. (C) Analysis of interactions within cohesin subunits. FRET experiments were performed with strains Y2598 (MATa/α SMC1hinge-YFP-C-CFP), Y2592 (MATa/α CFP-SCC1-YFP), Y2872 (MATa/α YFP-SCC3-CFP) and Y2865 (MATa/α CFP-PDS5-YFP).

Figure 4

Head–hinge interactions within the cohesin complex. (A) Pds5 interaction with both Scc1 and the Smc1 hinge. FRET was measured between fluorophore pairs as indicated, from left to right, in strains Y2531, Y2575, Y2532, Y2530, Y3264, Y2574, Y3275, Y2590, Y2706 and Y2705. Individual measurements are indicated as points, with mean shown as green and s.d. shown as blue lines. FRET_R was significantly different from the Pds5-YFP/Smc1-CFP pair at ^*P<0.01 and ^**P<0.0001, respectively, by Student's t-test. (B) Scc1-dependent interaction of Pds5 with Smc1. Extracts were prepared from haploid strains Y3223 (MATa SMC1-Pk3 GAL1-SCC1-HA6) and Y3210 (as Y3223, but PDS5-myc18) in the presence or 2.5 h after repression of Scc1, and co-immunoprecipitation of Smc1 with Pds5 analysed. (C) Direct interaction of the Smc1 head and hinge. Protein expression was induced in strains Y2318 (MATa GAL-SMC3hinge-Pk3 GAL-myc9-SMC1hinge), Y1823 (MATa GAL-SMC3hinge-Pk3), Y2319 (as Y2318, plus GAL-SMC1head-HA3) and Y1824 (as Y1823, plus GAL-SMC1head-HA3) for 2 h, and protein extracts prepared to analyse co-immunoprecipitation of the Smc1 head and hinge. An asterik indicates the cross-reacting immunoglobulin heavy chain.

Figure 5

Search for proximity of more than one cohesin complex in vivo. FRET measurements to analyse proximity of more than one Smc1 head, Smc3 head or Smc1 hinge, were conducted in strains Y3082 (MATa/α SMC1-CFP/SMC1-YFP), Y3083 (MATa/α SMC3-CFP/SMC3-YFP) and Y3228 (MATa/α SMC1hinge-CFP/SMC1hinge-YFP). The schematic representation illustrates geometries of possible cohesin interactions that have been tested and excluded by this analysis.