Red1 interacts with two 9-1-1 subunits. (A) Mapping of Red1 domains interacting with Mec3 and Ddc1 in two-hybrid assays. Mec3 binds Red1 at a region from amino acids 531–551 and Ddc1 at a region from amino acids 729–751. (Right) Two-hybrid interactions identified on selective media (−His) of fusions with an activating domain (AD) or DNA binding domain (BD) are shown. The white colony color is indicative of better growth. Images were taken after 3 d at 30 °C. (Left) Mec3 and Ddc1 binding sites are shown in gray in the diagram. Growth on complete media is shown as a control. (B) Comparison of the 9-1-1 binding domains in Red1 with the bona fide PIP box consensus core motif. Amino acids altered to alanine are shown in red. (C) Amino acid replacements in the Mec3 binding site of Red1 abolish Mec3 interaction, as indicated by two-hybrid assays (3 d at 30 °C). (D) Similarly, the amino acid replacement in the Ddc1 binding site of Red1 abolishes Ddc1 interaction.

9-1-1–Red1 interaction is essential for meiotic checkpoint signaling. (A) Ddc1 binding-deficient Red1 variant (Red1^−Ddc1) reverts meiotic checkpoint arrest of Δdmc1 deletion strains. Extracts of synchronously sporulating cells were made at the indicated times and probed by Western blot analysis for Zip1 and Pgk1 expression and in parallel for phosphorylated H2A (equivalent to mammalian γ-H2AX) and Rad52 SUMOylation as measures for checkpoint activation. The Y1083, CE571, and CE579 strains were used. (B) Meiotic (nuclear) divisions monitored by DAPI staining of chromosomal DNA. Samples from synchronously growing cultures were harvested at the indicated times and fixed in ethanol for subsequent DAPI staining. Symbols are identical to those used in C. (C) For germination assays, spores were isolated and zymolase-treated after 72 h in sporulation media and then plated on YDP plates at 30 °C, as described previously (43). For B and C, the Y1083, Y2031, CE571, and CE579 strains were used.

9-1-1–Red1 interaction is essential for SC formation. (A) SC formation assay. SCs were visualized using GFP-tagged Zip1 and spinning disk microscopy, as described in Fig. S3. Maturation of SCs was categorized in the indicated classes (early stage, diffuse, dot-like, pre-SCs, and full SCs). In the quantified assay, only full SCs and pre-SCs were distinguished and counted. (B) Full SCs and pre-SCs in WT and 9-1-1 binding-deficient Red1 variants (red1^−Mec3, red1^−Ddc1, and red1^{−Mec3,−Ddc1}) were monitored, as described in A. Shown are mean values from five independent experiments. For each time point, more than 100 cells were analyzed. The strains used were CE774, CE775, CE776, and CE777.

Red1 SUMOylation during meiosis mediates timing of SC formation. (A) SUMOylation of endogenous Red1. Diploid homozygous SK1 WT (CE382) or Δred1 (CE387) cells with an integrated version of ^HisSUMO were released into synchronous sporulation, and cell extracts were harvested after the indicated times. ^HisSUMO conjugates (monitored by Ni-NTA pull-down, followed by Western blotting using an anti-Red1 antibody) detect Red1 species carrying one, two, or more SUMO moieties. To control for pull-down efficiency, ^HisPol30 (PCNA)-expressing cultures were mixed with the meiotic cultures before lysis and pull-down, and ^HisPol30 was detected by Western blot analysis using an anti-Pol30 antibody (Bottom, Red1 input levels). (B) Red1 interacts with SUMO (Smt3) and Ubc9. For the two-hybrid assay, cells were transformed with respective activation domain (AD) and binding domain (BD) fusions and spotted on selective media and were grown for 3 d at 30 °C. (C) Red1 SUMOylation depends in vivo on the SUMO E3 ligase Zip3 (zip3 strain, CE566; done as in A; Lower, Red1 input levels). (D) Identification of SUMO-acceptor sites in Red1 using DF5 cells expressing ^HisSUMO and ^BDRed1^531–827, in which Red1 sequences were derived from WT Red1 or Red1 variants carrying KR exchanges (KR1, KR2, KR; defined in E). SUMO conjugates were isolated by Ni-NTA pull-down from lysates and detected by Western blotting using anti-BD monoclonal antibodies (Santa Cruz) (Lower, Red1 input levels). (E) Diagram of Red1 indicating a lysine-rich (K-rich) region (amino acids 569–590). Red1 variants harboring KR replacements of all lysines within regions of amino acids 569–577 (designated KR1), amino acids 579–590 (designated KR2), and amino acids 569–590 (designated KR) are indicated. (F) SUMOylation of endogenous Red1^KR. Ni-NTA pull-down of SUMO conjugates from lysates of SK1 strains (8 h after induction of sporulation) expressing Red1 WT (CE659) or the Red1^KR (CE662) variant from the genome using RED1 promoter and terminator elements. Western blot analysis using an anti-Red1 antibody. Control of pull-down efficiency as in A (^HisPol30). (Middle) Red1 input levels are shown. (G) SIM-containing C-terminal region of Zip1 specifically binds SUMOylation-proficient WT Red1 but not Red1^KR. Two-hybrid interactions of a C-terminal fragment of Zip1 (amino acids 846–875) with a Red1 fragment (amino acids 531–827) derived from WT Red1 or Red1^KR identified on selective media (−His). (H) Full SC formation in WT (CE774) and red1^KR mutant (CE778) was monitored as described in Fig. 3A and Fig. S3. Shown are mean values from six independent experiments. For each time point, more than 100 cells were analyzed.