Spo13 regulates cohesin cleavage

Abstract

A key aspect of meiotic chromosome segregation is that cohesin, the protein complex that holds sister chromatids together, dissociates from chromosome arms during meiosis I and from centromeric regions during meiosis II. The budding yeast protein Spo13 plays a key role in preventing centromeric cohesin from being lost during meiosis I. We have determined the molecular basis for the metaphase arrest obtained when SPO13 is overexpressed during the mitotic cell cycle. Overexpression of SPO13 inhibits anaphase onset by at least two mechanisms. First, Spo13 causes a transient delay in degradation of the anaphase inhibitor Pds1. Second, Spo13 inhibits cleavage of the cohesin subunit Scc1/Mcd1 or its meiosis-specific homolog, Rec8, by the separase Esp1. The finding that Spo13 did not prevent cleavage of another Esp1 substrate, Slk19, suggests that overexpression of SPO13 is sufficient to prevent cohesin cleavage by protecting specific substrates from separase activity.

Figure 1

Overexpression of SPO13 causes cells to arrest in metaphase. (A) Wild-type (▪, A940) and GAL–SPO13 (▴, A1813) cells were arrested in G₁ with α-factor (5 μg/mL) in YEPR (YEP + 2% raffinose). After 1 h, galactose was added to 2% to induce SPO13 expression. Cells were then released into medium containing galactose but lacking pheromone. The percentage of cells with metaphase (left graph) or anaphase (right graph) spindles was determined at the indicated times. (B,C) Wild-type (▪, A1951); TET–SPO13 (▴, A4318); rad9Δ, mad1Δ (○, A5309); and rad9Δ, mad1Δ, TET–SPO13 (⋄, A5310) cells containing a PDS1–HA fusion were grown in YEPR containing doxycycline (5 μg/mL) to inhibit SPO13 expression. Cells were then washed to remove the doxycycline and arrested in G₁ with α-factor (5 μg/mL) for 4 h, followed by release into YEPR lacking pheromone and doxycycline. After 100 min, when cells had budded, 5 μg/mL α-factor was added to prevent entry into the second cell cycle. The total amount of Pds1, Clb2, and Sic1 (B), as well as the percentage of cells with metaphase (C, left graph) or anaphase (C, right graph) spindles were determined at the indicated times. Kar2 was used as a loading control in Western blot analyses. (D) GAL–SPO13 (▴, A1767) and GAL–SPO13 pds1Δ (⋄, A1801) cells were grown as described in A, and the percentage of cells with metaphase (left graph) or anaphase (right graph) spindles was determined.

Figure 2

SPO13 inhibits Scc1/Mcd1 cleavage. (A) mad1Δ (▪, A928); mad1Δ, GAL–SPO13 (▴, A1796); mad1Δ, mcd1-1 (○, A4264); and mad1Δ, GAL–SPO13, mcd1-1 (⋄, A5413) cells were grown as described in Figure 1A except cells were released into YEPR medium containing galactose at 37°C. The percentage of cells with metaphase (left graph) or anaphase (right graph) spindles was determined at the indicated times. (B) Wild-type (▪, A3160) and TET–SPO13 (▴, A4003) cells containing an SCC1–18MYC fusion were grown as described in Figure 1B. The percentage of cells with metaphase (left graph) or anaphase (right graph) spindles was determined at the indicated times. The total amount of Scc1–Myc was determined by Western blot analysis. Cdc28 was used as a loading control. (C) TET–SPO13, GAL–TEV, SCC1–TEV268–HA (▪, A3475) and TET–SPO13 (▴, A3382) cells were grown in YEPR containing doxycycline (5 μg/mL). Cells were then washed and grown for 2 h in YEPR lacking doxycycline to induce SPO13 expression. Then 5 μg/mL α-factor was added to arrest cells in G₁, followed by release into YEPR lacking pheromone and doxycycline. After 110 min, when cells were arrested in metaphase, the cultures were split and galactose was added to 2% (open symbols) or glucose to 2% (closed symbols) to induce or inhibit the expression of the TEV protease, respectively. The percentage of cells with metaphase (left graph) and anaphase spindles (right graph) was determined at the indicated times. The total amount of Scc1–HA and Clb2 for strain A3475 was determined by Western blot analysis. Cdc28 was used as a loading control.

Figure 3

Spo13 inhibits cleavage of Rec8. (A,B) pSCC1–REC8–13MYC (▪, A5468) cells were grown as described in Figure 1B except that after release from α-factor arrest, the culture was split and 15 μg/mL nocodazole was added to one-half of the cells. After 110 min, when cells had budded, 5 μg/mL α-factor was added to prevent entry into the second cell cycle. The percentage of cells with metaphase (A, left graph) or anaphase (A, right graph) spindles and the total amount of Rec8–Myc, Clb2, and Sic1 were determined at the indicated times. (B) Cdc28 was used as a loading control in Western blot analyses. (C,D) pds1Δ, pSCC1–REC8–13MYC (▪, A5635) and pds1Δ, pSCC1–REC8–13MYC, TET–SPO13 (▴, A5645) cells were grown as described in Figure 1B, and the percentage of cells with metaphase (C, left graph) or anaphase (C, right graph) spindles was determined at the indicated times. (D) The total amount of Rec8–Myc, Clb2, and Sic1 was determined by Western blot analysis. Cdc28 was used as a loading control.

Figure 4

Spo13 does not inhibit cleavage of Slk19. pds1Δ (▪, A4188) and pds1Δ, TET–SPO13 (▴, A4187) cells containing SCC1–3HA and SLK19–13MYC fusions were grown as described in Figure 1B to determine the percentage of cells with metaphase (A, left graph) or anaphase (A, right graph) spindles. (B) The total amount of Scc1–3HA, Slk19–13MYC, and Clb2 was determined by Western blot analysis. Clb2-associated kinase activity was determined using Histone H1 as a substrate. At the 0 time point, three forms of Slk19–13MYC are visible. The upper band is full-length Slk19, which becomes phosphorylated as cells progress through the cell cycle. The bottom band corresponds to the C-terminal cleavage product of Slk19; and the middle band is the phosphorylated form of the cleavage product.