Red1 binds Smt3 chains F-M Lin et al Figure 6 The Red1–Smt3 chain interaction is essential for synaptonemal complex (SC) assembly. (A) The two-hybrid interaction between Zip1C and Red1C. Zip1C represents the C-terminal globular domain (residues 849–875). Zip1C3N 3R is incompetent in Smt3 binding because it contains three point mutations (E862R, D863R and Q864R) (Cheng et al, 2006). (B) Western blot time-course analysis of V5-Smt3 conjugates in the indicated yeast strains using anti-V5 antibody. Arp7 was used as a sample loading control. High steady-state levels of Smt3 chain conjugates were detected in the sporulating ulp2D and zip3D cells. The steady-state level of Smt3 conjugates was higher in the wild-type cells than in the zip1D, red1D and zip1Dzip3D cells. These results indicate that Red1 and Zip1 are required for the stable accumulation of Smt3 conjugates. Moreover, all of the examined cells, except ulp2D, exhibited a similar steady-state level of monomeric Smt3 proteins, indicating that Ulp2 likely acts together with at least one other new protein to maintain the concentration of Smt3 monomers in vivo. (C, D) Representative images of surface nuclei spreads for the indicated meiotic cells stained with DAPI (blue), anti-V5 antibody (for V5-Red1), anti-Myc antibody (for His6– Myc–Smt3) and anti-Zip1 antibody. The black bar represents 5 mm. (E) The time course of SC formation was determined by assigning spread nuclei into four classes according to different Zip1 staining patterns: full SC, partial SC, Zip1 foci and polycomplex. gous chromosomes would be greatly diminished during such rapid movements. The Smt3 chain relay mechanism may also explain the roles of centromeres in SC polymerisation. An early chroma- tin immunoprecipitation study showed that Red1 is highly enriched in the chromosome arms and less abundant at the centromere (Blat et al, 2002). During or before early zygo- tene, centromeres may act as SC initiation sites (Tsubouchi and Roeder, 2005; Tsubouchi et al, 2008) because the non- Zip3 SUMO E3 ligases not only produce Smt3 conjugates (e.g., Top2) at centromeres for NHCC (Tsubouchi and Roeder, 2005; Cheng et al, 2006) but they also generate ‘diffusible’ Smt3 chains for neighbouring Red1 to mediate SC polymer- isation. These non-Zip3 ligases are then downregulated after zygotene, and the centromeric Smt3 conjugates are desumoy- lated by Ulp2 (Cheng et al, 2006). The centromere then & 2010 European Molecular Biology Organization serves as a barrier for Zip3-dependent SC polymerisation during pachytene (Henderson and Keeney, 2005) because centromeres contain fewer Red1 proteins. Our results also revealed that the Red1–Smt3 chain inter- action is required for Mec1–Tel1 mediated Hop1 phosphor- ylation (Carballo et al, 2008). As the two SIM mutants examined in this study were not defective in Hop1 chromoso- mal localisation (Supplementary Figure S2) or the Red1– Hop1 interaction (Supplementary Table S2 and Figure S3), we speculate that Smt3 chains may recruit Tel1 and Mec1 kinases to DSB sites for Hop1 phosphorylation. We are at present investigating whether these two kinases or their binding partners (e.g., Tel2 and Ddc2–Lcd1–Pie1) (Paciotti et al, 2000; Rouse and Jackson, 2000; Wakayama et al, 2001; Nakada et al, 2005; Anderson et al, 2008) contain a puta- tive SIM. The EMBO Journal VOL 29 | NO 3 | 2010 593