Phosphorylation of soluble Scc1 by overexpressed Polo makes its cleavage comparable to chromatin-bound Scc1. (A) Hyperphosphorylation of soluble Scc1 in strain Y1585 (MATa SCC1-HA3 CDC5 GAL-CDC5) after induction of Polo (encoded by CDC5) in metaphase-arrested cells. (B) Soluble and chromatin-bound cohesin was prepared 1 h after Polo induction, and its cleavage was compared to soluble and chromatin-bound cohesin from uninduced cells.

Scc1 in cohesin that was released from chromatin by DNase I treatment is cleaved at a similar rate to chromosomal Scc1. (A) Cleavage of chromatin-released Scc1-HA (rel) was compared in the same reaction with soluble (sol) or chromatin-bound (cb) Scc1-myc. (B) Cleavage of chromatin-released Scc1-myc was compared with soluble and chromatin-bound Scc1-HA. (C) Summary of rate constants obtained in parallel reactions for cleavage of Scc1 in chromosomal, chromatin-released, and soluble cohesin. All reactions contained the same volumes of soluble cell fractions and separase-enriched extract. Average rate constants are given together with error bars indicating the standard deviation (n=3 for chromatin-bound and soluble Scc1-HA and Scc1-myc, n=2 for chromatin-released Scc1).

Fast cleavage of recombinant, phosphorylated Scc1 is partly due to higher affinity for separase. (A) Phosphorylated recombinant Scc1 (Scc1-P) is cleaved faster than phosphatase-treated Scc1 (Scc1). Recombinant Scc1 was incubated with separase and purified by chitin affinity chromatography from strain Y334. Cleavage was analysed by quantitative Western blotting, and relative amount of the cleavage product in each reaction is given. In one reaction, separase was phosphatase treated, denoted by ^*, before incubation with Scc1. (B) Separase binding to Scc1. Phosphorylated and phosphatase-treated recombinant Scc1 was bound to catalytically inactive separase (C1531A) that was affinity purified on chitin beads. Bound protein was analysed by quantitative Western blotting; binding is indicated relative to the respective input. A reaction in which separase was phosphatase treated is also included, denoted by ^*.

Chromatin provides a Polo-rich environment. (A) Polo in the chromatin-bound fraction. Whole extract (we), chromosomal (cb), and soluble (sol) cellular fractions were prepared from strain Y1570 (MATa SCC1-HA6 CDC5-myc18); Scc1 and Polo were detected by Western blotting. (B) Polo associates with chromatin independently of cohesin. Scc1 was depleted from strain Y1571 (MATa GAL-SCC1-myc18 CDC5-HA6) and cells were arrested in metaphase by nocodazole treatment. Chromatin binding of Scc1 and Polo was analysed by chromosome spreading, and levels of both proteins in soluble and chromatin-bound fractions were confirmed by Western blotting. (C) Cohesin binds chromatin independently of Polo. Cells were arrested by nocodazole treatment after depletion of Polo in strain Y8646 (MATa GAL-CDC5 SCC1-HA6). Polo depletion was confirmed by the fast mobility of hypophosphorylated Scc1. Cohesin binding to chromosomes was analysed by chromosome spreading and cellular fractionation.

Experimental outline to compare the rates of Scc1 cleavage in chromatin-bound versus soluble cohesin. Whole-cell extracts of nocodazole-arrested cells were fractionated by centrifugation through a 30% sucrose cushion into chromatin-bound and soluble proteins. Chromatin and soluble fractions containing differentially tagged Scc1 were mixed and cell extract containing overexpressed separase was added. The two reciprocal reactions were incubated at 25°C and aliquots retrieved at 1 min intervals for analysis by quantitative Western blotting.

Scc1 in chromosomal cohesin is cleaved at a faster rate than Scc1 in soluble cohesin. (A) Western blot analysis of the two Scc1 cleavage reactions described in Figure 1. Reaction 1 compares cleavage of chromatin-bound Scc1-HA with soluble Scc1-myc, and reaction 2 compares cleavage of chromatin-bound Scc1-myc with soluble Scc1-HA. The strains were Y58 (MATa SCC1-myc18) and K8869 (MATa SCC1-HA6). Separase overexpression was in strain Y334 (MATa GAL-flag-ESP1-CBD). (B) Quantification of remaining full-length Scc1 in the chromatin-bound (cb) and soluble (sol) fraction over time. Bands on the Western blot in (A) were quantified using an IRDye800 coupled secondary antibody and an Odyssey fluorescence scanner (LI-COR). The graph was fitted with a first order reaction (bold line), and the first order rate constant k₁ was derived.

Scc1 in chromatin-bound cohesin is hyperphosphorylated compared to Scc1 in soluble cohesin. (A) Schematic representation of the 10 known Polo phosphorylation sites in Scc1. The two separase cleavage sites are indicated by arrows. (B) Migration of chromatin-bound (cb) and soluble (sol) Scc1, and the effect of mutations replacing serines S175 and S263 (S175,263A), or all 10 potential phosphoserine residues to alanine (10 × S → A). Chromatin-bound and soluble fractions of nocodazole-arrested strains Y1287 (MATa GAL-SCC1-myc18 SCC1promoter-SCC1-HA3), Y1296 (MATa GAL-SCC1-myc18 SCC1promoter-SCC1(S175,263A)-HA3), and Y1288 (MATa GAL-SCC1-myc18 SCC1promoter-SCC1(S175,183,194,263, 273,276,325,374,389,497A)-HA3) were analysed by Western blotting. Remaining slow migration of the chromatin-bound 10 × S → A mutant was resolved after treatment with λ-phosphatase. (C) Response of the Scc1 cleavage rate to phospho-site mutations and to dephosphorylation by λ-phosphatase. Scc1-HA cleavage was analysed in parallel reactions using chromatin-bound or soluble cohesin preparations.

Phosphorylation accelerates cleavage of chromosomal Scc1 in vivo. (A) Faster cleavage of chromatin-bound Scc1 in vivo depends on phosphoserine residues. Strains Y1447 (MATa GAL-CDC20 PDS1-myc18 SCC1promoter-SCC1-HA3) and Y1449 (MATa GAL-CDC20 PDS1-myc18 SCC1promoter-SCC1(S175,183,194,263, 273,276,325,374,389,497A)-HA3) were arrested in metaphase by depleting Cdc20 and released into synchronous anaphase at 16°C. Soluble and chromatin-bound Scc1 was separated in samples taken at the indicated time points, and full-length Scc1 was quantified. Note that cleaved cohesin is released from chromatin, thus the Scc1 cleavage product in the soluble fraction originates partly from chromatin. (B) 10 × S → A mutant Scc1 dissociates from chromatin later and delays elongation of the anaphase spindle. As (A), but degradation of securin and spindle elongation were analysed by indirect immunofluorescence, and Scc1 binding to chromatin was visualised on chromosome spreads. (C) 10 × S → A mutant Scc1 confers increased chromosome loss. Strains Y1305 (MATa CFIII (CEN3.L.YPH278) URASUP11 SCC1promoter-SCC1-HA3) and Y1306 (MATa CFIII (CEN3.L.YPH278) URASUP11, SCC1promoter-SCC1-S175,183,194,263, 273,276,325,374,389,497A-HA3) were grown on medium lacking uracil to maintain a marker chromosome, and plated on rich medium. Half red sectored colonies were counted, indicative of chromosome loss in the first division after plating. Chromosome loss in the strain containing 10 × S → A Scc1 is larger than in wild type with a confidence level greater than 93.75%.