Fig. 2: S. pombe Sor1 is involved in sister chromatid cohesion and its conserved residues are important for Sor1 function and association with cohesin.

a sor1Δ cells show a weak cohesion defect which is partially suppressed by wpl1Δ. Wild type and sor1Δ haploid cells expressing cen2-GFP were fixed and stained with antibodies against tubulin and GFP and sister chromatid cohesion was analysed in metaphase cells. Nuclei were visualized by Hoechst staining. Means +/− standard deviations are shown. Unpaired t-test was performed for statistical analysis (**p < 0.01; ns—not significant). b Negative synthetic growth interaction in eso1ts sor1Δ and mis4ts sor1Δ double mutants are associated with chromosome segregation defects. Wild type, sor1Δ, eso1-G799D (eso1-ts), eso1-G799D sor1Δ, mis4-242 (mis4-ts) and mis4-242 sor1Δ haploid cells expressing cen2-GFP were fixed and stained with antibodies against tubulin and GFP. Nuclei were visualized by Hoechst staining. Samples were examined under the fluorescence microscope, and segregation of chromosome 2 marked by cen2-GFP was scored in late anaphase cells. Lagging chromosomes were identified as Hoechst-staining bodies between the poles of the spindle in late anaphase. Means +/− standard deviations are shown. Unpaired t-test was performed for statistical analysis (*p < 0.05; **p < 0.01). c Pds5-Myc co-immunoprecipitates with Sor1-Pk. Protein extracts were prepared from cycling wild-type cells and cells expressing Sor1-Pk, Pds5-myc or both Sor1-Pk and Pds5-Myc, as indicated. Proteins bound to anti-V5 agarose beads, which bind the Pk tag on Sor1, were analysed for Pds5-Myc by Western blotting using anti-Myc antibody. The experiment was repeated twice with similar results. d Psm3-GFP co-immunoprecipitates with Sor1-Pk and this interaction is weakened by mutating conserved Sor1 residue D303. Protein extracts were prepared from cycling wild-type cells and cells expressing Sor1-Pk, Psm3-GFP or both Sor1-Pk and Psm3-GFP, as indicated. Proteins bound to anti-GFP agarose beads, which bind the GFP tag on Psm3, were analysed for Sor1-Pk by Western blotting using anti-V5 antibody. Mutant protein Sor1-D303A-Pk co-immunoprecipitated with the Psm3-GFP protein less efficiently, as compared to wild-type Sor1-Pk. The experiment was repeated twice with similar results. e The four conserved residues in the Sororin domain are important for the Sor1 function. Strains with the indicated mutations were grown on YES medium for one day. Serial dilutions were spotted onto YES plates and incubated for 3 days at 25 °C or 30 °C. While expression of a wild type Sor1 rescued the growth defect of the eso1-G799D sor1Δ double mutant (eso1-ts sor1-wt), mutant Sor1 proteins carrying F299A, V302A, D303A or Y305A substitutions did not rescue the growth defect of eso1-G799D sor1Δ double mutants (eso1-ts sor1-F299A, eso1-ts sor1-V302A, eso1-ts sor1-D303A, eso1-ts sor1-Y305A).