Controlled access model for sister chromatid cohesion. Shown are Pds5p (hatched ovals), the cohesin complex (open circles), SUMO (black ovals), and Nfi1p and Smt4p (open ovals). Ubc9p binds SUMO and transfers it to targets, but Nfi1p provides target specificity. For simplicity, we show only Nfi1p.

SMT4 suppresses pds5 mutant temperature sensitivity. (A) Effect of high copy SMT4 (2μ URA3 vector) on the temperature-sensitive phenotype of pds5 mutants. Haploid strains pds5-1 (VG986-5B), pds5-2 (VG987-5C), and pds5-3 (VG988-1C) containing 2μ PDS5 (pVG175), 2μ (pRS202), or 2μ SMT4 (pTH5) grown to saturation at 23°C in SC-URA liquid, plated in 10-fold serial dilutions on YEPD, and incubated for 72 h at 23 or 37°C. (B) Effect of low copy SMT4 (CEN URA3 vector) on the temperature-sensitive phenotype of pds5 mutants. Haploid pds5-1 (VG986-5B), pds5-2 (VG987-5C), and pds5-3 (VG988-1C) strains containing CEN PDS5 (pVG282), CEN (YCplac33), or CEN SMT4 (pTH4) grown and plated as described in A.

Modulation of Pds5p sumoylation by SUMO pathway genes. Pds5p sumoylation determined by IP Western blot as described in Fig. 4 A. (A) Effect of SMT4 overexpression. Haploids containing pGAL-SMT4 (VG2525-2B) or pGAL (VG2524-1A) were grown in YEP raffinose at 23°C, and then galactose was added for 1 h. Aliquots from raffinose- and galactose-grown cells were processed for IP Western blot analysis of Pds5p. SUMO conjugation (top blot) and Pds5p (bottom blot). IPs from two cultures of pGAL-SMT4 cells are shown. (B) Pds5p sumoylation in smt4 mutants. Haploids smt4 PDS5-6MYC (VG2465-4D), PDS5-6MYC (VG2066-7B), and smt4 PDS5 (VG2463-1D) were grown in YEPD at 23 or 37°C, and then Pds5p was immunoprecipitated. IP Western blots showing Pds5p sumoylation (top blot) and Pds5p (bottom blot). (C) Effect of SUMO E3 and isopeptidases on Pds5p sumoylation. Pds5p was immunoprecipitated from extracts of asynchronous haploid PDS5-6MYC (VG2066-7B) cells containing 2μ SMT4 (pTH5), 2μ ULP1(pPM237), 2μ NFI1 (pPM353), or 2μ vector (YEplac195). IP Western blots showing Pds5p sumoylation (top blot) and Pds5p (bottom blot).

SMT4 overexpression suppresses precocious sister chromatid dissociation in pds5 cells. Mutant pds5-2 cells bearing either pGAL-SMT4 (VG2445-5B) or pGAL (VG2446-6A) grown as described in Fig. 6 were fixed to monitor cohesion near URA3. (A) Percentage of pds5 cells with separated sister chromatids. The number of cells with one or two GFP signals was determined. Cells with separated sisters (two GFP signals) were plotted as a percentage of total cells. Three independent experiments were performed, and 200–300 cells at each time point in each experiment were scored to generate data and error bars. (B) Relative distance between separated sisters. In cells containing two GFP signals, the distance between GFP signals was scored as near (separated by less than one bud length) and far (separated by more than one bud length). Three independent experiments were performed. (C) Comparison of sister separation in pds5 cells overexpressing SMT4 and wild-type cells. Mutant pds5-2 cells bearing pGAL-SMT4 (VG2445-5B) and wild-type cells (VG2452-7A) were grown and scored for sister separation as described above.

Specificity of SMT4 suppression. (A) Effect of high copy SMT4 (2μ URA3 vector) on the temperature-sensitive phenotype of mutants defective in chromosome structure or cell cycle progression. Haploids pds5-1 (VG986-5B), pds5-3 (VG988-1C), mcd1-1 (VG985-7C), mcd1-73/scc1-73 (K5832), smc1-2 (VG1360-7C), smc1-259 (K6013), smc3-42 (K5824), scc2-4 (K5828), smc2-8 (VG2029-7B), top2-4 (VG2014-4D), pds1-2 (VG971-1A), and esp1-1 (2788) containing 2μ SMT4 (pTH5) and either 2μ (pRS202) or 2μ (YEplac195) were grown to saturation at 23°C in SC-URA liquid, serially diluted fivefold (first well) and 10-fold (second well) on YEPD plates, and were then incubated at 23 or 37°C for 72 h. (B) Effect of high copy plasmids bearing genes in the SUMO pathway on the temperature-sensitive phenotype of pds5 mutants. Haploid pds5-1 (VG986-5B), pds5-2 (VG987-5C), and pds5-3 (VG988-1C) cells bearing high copy 2μ plasmids SMT4 (pTH5), ULP1(pPM237), NFI1 (pPM353), or vector only (YEplac195) were grown and plated as described in Fig. 2.

Characterization of cell cycle and cohesion defects of pds5 cells. Wild-type (VG2450-7A and VG2390-37A) and pds5-2 (VG2456-5C and VG2416-12A) haploids released from S phase (HU arrest) at 37°C. (A) Percentage of cells with two GFP signals. The number of cells with two GFP signals was determined in HU-arrested cells (t = 0) and at various times after release from arrest, and were then plotted as the percentage of total cells (see Materials and methods). (B) DNA content of cells by FACS^® analysis. (C) Pds1p levels. Wild-type (VG2450-7A) and pds5-2 (VG2456-5C) cells subjected to Western blot using anti-HA (Pds1-3HA) and anti-β tubulin (Tub2) antibodies (see Materials and methods). (D) Micrographs of cells with two GFP signals after release from HU arrest at 37°C. Chromosomal DNA (DAPI) and URA3 locus (GFP) are shown. Bars, 5 μm. Data were generated from two independent experiments for all figures. For A, 100–200 cells were scored at every time point to generate error bars.

Sumoylation of Pds5p. (A) IP Western blot of Pds5p from asynchronous cells. PDS5-6MYC (VG2066-7B), PDS5-12MYC (VG2067-2B), and PDS5 (VG982-6A) haploids were grown in YEPD at 23°C, and total protein extracts were made. Pds5p was immunoprecipitated using anti-MYC antibodies, and SUMO conjugation was detected by Western blot using anti-SMT3 antibody (left gel). The position of two prominent Sumo bands is marked for the Pds5-6MYCp IP. The blot was stripped and probed with anti-MYC antibody to detect Pds5p (right gel). As a reference, the position where unsumoylated Pds5p (asterisk) would migrate is shown on the SUMO blot. (B–E) Synchronous populations of PDS5-6MYC (VG2066-7B) cells released from S phase arrest (HU). (B) IP Western blot of Pds5p. MYC-tagged Pds5p was immunoprecipitated, then SUMO and Pds5p were detected as described in A. Pds5p sumoylation (top gel). Two prominent Sumo bands are marked, as is the position where unsumoylated Pds5p (asterisk) migrates. The blot was stripped and probed to detect Pds5p (bottom gel). (C) Western blot of Pds5p. Total protein extracts were analyzed using anti-MYC (Pds5-6MYC) or anti-tubulin (Tub2) antibodies. (D) DNA content of cells by FACS^® analysis. (E) Relative sumoylation of Pds5p. The SUMO/Pds5p ratio was determined at each time point by densitometry of Western blot in B. (F and G) Pds5p sumoylation in arrested cells. Cells bearing PDS5-6MYC were arrested in G1 phase (αF), S phase (HU), metaphase (Nz) using haploid VG2066-7B, or telophase (cdc15) using haploid VG2424-4C. (F) IP Western blot to detect Pds5p sumoylation. (G) Relative sumoylation of Pds5p. The SUMO/Pds5p ratio was determined by densitometry.

Effect of SMT4 overexpression on viability and cell cycle progression of pds5 cells. Mutant pds5-2 cells bearing either pGAL-SMT4 (VG2445-5B) or pGAL (VG2446-6A) and wild-type cells (VG2452-7A) were grown in YEP raffinose at 23°C, were arrested in S phase, galactose was added, and then cells were transferred to 37°C (t = 0). Cells were released from arrest at 37°C, and aliquots were processed every 15 min for FACS^® analysis or were plated on YEPD to determine viability. (A) Relative viability. Percentage of cell viability was determined at each time point and normalized to viability in HU-arrested cells. Three independent experiments were performed to generate data and error bars. (B) DNA content of cells by FACS^® analysis. (C) Pds1p levels. Mutant pds5-2 cells bearing PDS1-3HA and either pGAL-SMT4 (VG2485-3B) or pGAL (VG2486-14A) were grown as described above. Western blots of total proteins using anti-HA (Pds1-3HA) or anti-β tubulin (Tub2) antibodies. One of two independent experiments is shown. (D) Time of anaphase execution in wild-type cells. Large budded cells from haploid strain VG2452-7A in A were scored for DNA morphology to determine when anaphase occurs. 100 cells at each time point from three independent experiments were scored to generate data and error bars.