Mes1 controls the meiosis I to meiosis II transition by distinctly regulating the anaphase-promoting complex/cyclosome coactivators Fzr1/Mfr1 and Slp1 in fission yeast

Abstract

Meiosis is a specialized form of cell division generating haploid gametes and is dependent upon protein ubiquitylation by the anaphase-promoting complex/cyclosome (APC/C). Accurate control of the APC/C during meiosis is important in all eukaryotic cells and is in part regulated by the association of coactivators and inhibitors. We previously showed that the fission yeast meiosis-specific protein Mes1 binds to a coactivator and inhibits APC/C; however, regulation of the Mes1-mediated APC/C inhibition remains elusive. Here we show how Mes1 distinctively regulates different forms of the APC/C. We study all the coactivators present in the yeast genome and find that only Slp1/Cdc20 is essential for meiosis I progression. However, Fzr1/Mfr1 is a critical target for Mes1 inhibition because fzr1Δ completely rescues the defect on the meiosis II entry in mes1Δ cells. Furthermore, cell-free studies suggest that Mes1 behaves as a pseudosubstrate for Fzr1/Mfr1 but works as a competitive substrate for Slp1. Intriguingly, mutations in the D-box or KEN-box of Mes1 increase its recognition as a substrate by Fzr1, but not by Slp1. Thus Mes1 interacts with two coactivators in a different way to control the activity of the APC/C required for the meiosis I/meiosis II transition.

FIGURE 1:

Roles for fission yeast APC coactivators in meiotic progression. (A) Schematic diagrams representing the domains of five Fizzy/Cdc20 family APC/C coactivators in fission yeast. All of them share a C-box motif (C in the black box) and seven tandem repeats of WD40 domain (7x WD40) and terminate at either IR, LR, or VR dipeptides. (B) Synchronized meiosis by pat1-114 inactivation in WT, Puhp1-HA-ste9, fzr2Δ, and fzr3Δ diploid cells. Meiotic progression was examined microscopically for the number of nuclei in a cell. Protein levels were analyzed by immunoblotting using specific antibodies at indicated time points. (C) Same as B, but meiotic progression in WT, fzr1Δ, and slp1-NF410 mutant diploid cells upon pat1-114 inactivation was examined.

FIGURE 2:

Fzr1/Mfr1 activates APC/C in anaphase I in the absence of Slp1 activity. (A) Synchronized meiosis in WT, slp1-NF410, and K0-mes1 diploid cells. Meiotic progression was examined microscopically for the number of nuclei per cell as well as the number of short spindles stained by anti-tubulin antibody. Protein levels were analyzed by immunoblotting using specific antibodies. (B) As in Figure 1B, meiotic progression in WT, slp1-NF410, and slp1-NF410fzr1Δ diploids was monitored.

FIGURE 3:

Mes1 inhibits Fzr1/Mfr1 during meiosis I. Meiosis was induced by thermal inactivation of Pat1, and meiotic progression was monitored in WT, mes1Δ, mes1Δslp1-NF410, and mes1Δfzr1Δ mutant diploid cells. Samples were examined microscopically for the number of nuclei in a cell to monitor progression through meiosis and analyzed by immunoblotting using specific antibodies at indicated time points.

FIGURE 4:

Roles for coactivators in Mes1 turnover during meiosis. (A) pat1-114–induced synchronization of meiosis was performed in WT, fzr1Δ, slp1-NF410, and slp1-NF410fzr1Δ mutant diploid strains expressing HA-tagged Mes1 under the native promoter. HA-Mes1 levels were analyzed by immunoblotting using anti-HA antibody. The levels of HA-Mes1 did not show any change by fzr1Δ but were significantly increased by slp1-NF410 mutations. (B) HA-tagged Mes1 levels were analyzed in synchronized meiosis in WT, Puhp1-HA-ste9, fzr2Δ, and fzr3Δ strains using anti-HA antibody.

FIGURE 5:

APC/C-dependent ubiquitylation and destruction by different coactivators. (A) In vitro ubiquitylation assays using S. pombe APC/C and coactivators. ³⁵S-labeled Cut2 and Cdc13 were used as substrates. Samples were taken at indicated time points and analyzed by SDS–PAGE followed by autoradiography. (B) The intensity of the signals corresponding to the ubiquitinated substrates in A was quantified and presented in the graph. (C) Destruction assays of Cdc13 in S. pombe APC/C, E1, E2, and coactivators supplemented Xenopus egg extracts from which endogenous APC/C and Fizzy/Cdc20 had been depleted.

FIGURE 6:

Fzr1/Mfr1 cannot activate APC/C for Mes1 ubiquitylation and destruction. (A, B) In vitro ubiquitylation assay of Mes1 was performed as in Figure 5A except using Mes1 as a substrate. The intensity of ubiquitylated Mes1 signals was quantified and shown in the graph. (C) Destruction assays were performed as in Figure 5C apart from using Mes1 as a substrate.

FIGURE 7:

Different relationship between Mes1 and Slp1 or Fzr1/Mfr1 toward Mes1 ubiquitylation and destruction. (A) In vitro binding between coactivators (TK15-tagged Slp1 or TK15-tagged Fzr1) and Mes1 was analyzed. The coactivators and ³⁵S-labeled Mes1 were incubated together, and the activators were immunoprecipitated using anti-TK15 beads. Three dilutions for each immunoprecipitate were analyzed by SDS–PAGE, and copurified Mes1 was determined by autoradiography. KEN-box mutant (Km), D-box mutant (Dm), and KEN-box and D-box double mutant (DK) of Mes1 were used as controls. Similar amounts of WT Mes1 were bound to Slp1 and Fzr1/Mfr1 in a D-box– and KEN-box–dependent manner. (B) Slp1- or Fzr1/Mfr1-dependent ubiquitylation assays were performed using WT, KM, DM, and DK of Mes1 as a substrate. Dm or Km Mes1 protein was more efficiently ubiquitylated than WT Mes1 by S. pombe Fzr1/Mfr1–APC/C. (C) Destruction assays of Mes1 in Xenopus egg extracts from which endogenous APC/C was predepleted and S. pombe APC/C and Slp1 or Fzr1/Mfr1 were supplemented. Fzr1/Mfr1–APC/C destroyed Dm or Km more progressively than WT Mes1.

FIGURE 8:

Slp1, but not Fzr1/Mfr1, overcomes Mes1 inhibition via ubiquitylation. (A) In vitro ubiquitylation assays were performed with ³⁵S-labeled Cut2 as a substrate and Mes1 as an inhibitor. Samples were taken at indicated time points and analyzed by SDS–PAGE followed by autoradiography. Mes1 efficiently inhibited Cut2 ubiquitylation induced by Slp1, Ste9, Fzr1/Mfr1, and Fzr2. (B) Destruction assays were performed using Cdc13 as a substrate as described in Figure 5C except that the coactivator(s), Slp1 and/or Fzr1/Mfr1, and an inhibitor WT or K1-Mes1 were simultaneously added into the extracts. Samples were taken at the indicated time points and analyzed by SDS–PAGE followed by autoradiography. K1-Mes1 has an intact KEN-box in the construct. (C) A model for ubiquitylation-dependent regulation of Slp1 and Fzr1/Mfr1 by Mes1 in meiosis. Mes1 binds both Slp1 and Fzr1/Mfr1 and initially inhibits them as a competitive substrate. Slp1 is able to ubiquitylate Mes1 and escape from complete inhibition, whereas Fzr1/Mfr1 is kept inactive during anaphase I (meiosis I) due to its inability to ubiquitylate Mes1. In meiosis II, when Mes1 is ubiquitylated and degraded, both Slp1 and Fzr1/Mfr2 become active and complete anaphase II (meiosis II).