known to interact with itself, Hop1 and the protein phospha- tase, Glc7 (Bailis and Roeder, 2000; Woltering et al, 2000). Zip1 is the major structural component of the central region (Sym et al, 1993), The Zip1 proteins form head-to- head dimers connected by protein–protein interactions be- tween the central coiled-coil domains, and the C-terminal globular domain connects the central region to the AEs (Dong and Roeder, 2000). The SC initiation protein Zip3 is an E3 ligase for Smt3, the yeast small ubiquitin-like modiﬁer (SUMO). Post-translational modiﬁcations of SUMO control SC assembly through an interaction between the Zip1 C-terminal globular domain (residues 849–875) and the Zip3-dependent Smt3 conjugates along two AEs (Cheng et al, 2006). In the wild-type cell, Zip3 recruits Ubc9, the SUMO E2 enzyme, to meiotic chromosomes, but in the zip3D mutant, Ubc9 proteins are not associated with chromosomes (Hooker and Roeder, 2006). However, the Ubc9 proteins in mutant cells still catalyse the self-polymerization of Smt3 monomers to form Smt3 chains (Cheng et al, 2006). These chromosome-free Smt3 chains non-covalently associate with Zip1 proteins to form one or a few large proteinaceous aggregates known as polycomplexes (PCs). Zip1 stabilizes these chromosome-free Smt3 chains against the Ulp2 SUMO protease, resulting in a massive accumulation of Smt3 chains (Cheng et al, 2006). Other ZMM proteins (i.e., Zip2, Spo16) have been proposed to act after Zip3 as chaperone-like machinery to facilitate the perpendicular alignment of Zip1 proteins along two AEs during SC elongation. The meiotic chromosomes of these two zmm mutants can recruit Zip1, but are barely able to form long and ﬁne Zip1 lines between homologous chromosomes (Cheng et al, 2006). This observa- tion is consistent with a recent report that Zip2 functions in SC elongation is dependant upon SPO11 (Tsubouchi and Roeder, 2005; Tsubouchi et al, 2008). Spo11 generates DSBs, subsequently promoting Hop1 phosphorylation to ensure the close juxtaposition of two homologous chromosomes, by IH recombination, for SC elongation (Niu et al, 2005; Carballo et al, 2008). Zip1 also mediates pair-wise non-homologous centromere coupling (NHCC) before the zygotene stage (Tsubouchi and Roeder, 2005). Zip1 binds to centromeric Smt3 conjugates (e.g., Top2), which is mediated by non-Zip3 SUMO E3 ligases, Siz1, Siz2 or Mms21. These non-Zip3 E3 ligases are down- regulated after the zygotene stage. The NHCC is then de- stroyed by SUMO protease Ulp2 (Cheng et al, 2006). NHCC could lead to SC initiation at the centromeres. These centro- meric SCs are independent of SPO11 and ZIP3, which have major roles in the chromosome synapsis at non-centromeric locations. On the other hand, Spo11 and Zip2 are required for SC elongation at both centromeric and non-centromeric sites (Tsubouchi and Roeder, 2005; Tsubouchi et al, 2008). These two kinds of SCs are initiated differently but likely use the same mechanism for SC elongation. As described above, Spo11 is required for Hop1 phosphorylation, which ensures IH-recombination by repressing IS-recombination (Niu et al, 2005; Carballo et al, 2008). However, DSB-dependent Hop1 phosphorylation is not a prerequisite for SC initiation, because Zip1 and Zip3 can be recruited to meiotic chromo- somes in the absence of SPO11, for example, a previous study found that 67% of Zip3 foci were centromeric and 33% were non-centromeric (Tsubouchi et al, 2008). The function of DSB-dependent Hop1 phosphorylation at non-centromeric & 2010 European Molecular Biology Organization Red1 binds Smt3 chains F-M Lin et al locations is to closely juxtapose two homologous chromo- somes for SC initiation and elongation, whereas the function of DSB-independent NHCC at centromeres is to initiate SC assembly between two non-homologous chromosomes (Tsubouchi et al, 2008). Therefore, we reason that other non-Hop1 AE structural proteins may be responsible for Zip3- and Smt3-dependent SC initiation. In this study we found that, like Zip1, Red1 can bind Smt3 chains. The Red1–Smt3 interaction ﬁrst facilitated chromo- some recruitment of Zip1 and Zip3 for SC initiation, similar to the function of centromeric Smt3 conjugates in recruiting Zip1 and Zip3 proteins during NHCC (Tsubouchi and Roeder, 2005; Cheng et al, 2006; Tsubouchi et al, 2008). The second function of the Red1–Smt3 interaction was to promote Hop1 phosphorylation, which prevents IS-recombination to ensure IH-recombination. As a result, the two homologous chromo- somes are closely juxtaposed for Zip2 or other ZMM proteins to facilitate the perpendicular alignment of Zip1 proteins during SC elongation (Tsubouchi and Roeder, 2005; Cheng et al, 2006; Tsubouchi et al, 2008). Results Red1 binds Smt3 chains but not to Smt3 monomer A previous genome-wide two-hybrid screen suggested that Red1 might interact with Smt3 (Ito et al, 2001). However, it was unclear if the Red1–Smt3 two-hybrid interaction was because of a direct physical interaction between Red1 and Smt3 monomer, Smt3 polymeric chain or conjugate. Moreover, this two-hybrid interaction may have indirectly resulted from post-translational Smt3 modiﬁcation of Red1 itself or some other Red1 binding protein. The latter hypoth- esis is not favoured for the following reasons: ﬁrst, it is inconsistent with the result that the zip3D mutant could form short SCs in the absence of the Zip3-dependent Smt3 modiﬁed AE proteins (Agarwal and Roeder, 2000; Hooker and Roeder, 2006); second, very little Red1 protein (o1%) is Smt3-modiﬁed in wild-type sporulating cells (Cheng et al, 2006, 2007). In this study, we identiﬁed two putative Smt3-interacting motifs (SIMs) in the C-terminal portion of Red1, viz. SIM-1 (ILESTTVID, residues 712–720) (Minty et al, 2000) and SIM-2 (ISII, residues 758–761) (Song et al, 2004). The Red1C protein (residues 611–827) displayed a strong two-hybrid interaction with Smt3. We also constructed three mutant proteins: Red1CS715R,T717R (referred to as Red1C2R), Red1CI758R and Red1CS715R,T717R,I758R (referred to as Red1C3R). The order of two-hybrid interaction with Smt3 for these four proteins was Red1C4Red1C2R4Red1CI758R4Red1C3R (Figure 1A), indicating that SIM-2 is better than SIM-1 at binding Smt3. Red1C did not exhibit a two-hybrid interaction with Smt3- DGG, a conjugation-incompetent Smt3 mutant lacking the C-terminal pair of glycines for E1-mediated Smt3 activation (for a review see Gill, 2004). In addition, Red1C and Smt3 induced a high level of two-hybrid interaction in smt3-allR reporter cells. However, Red1C and Smt3-allR exhibited a weaker interaction in the same reporter cells (Figure 1A). Smt3-allR is competent in the SUMO conjugation to all target proteins, including the wild-type Smt3. However, Smt3-allR cannot form a polymeric chain because the nine lysine residues in Smt3 were replaced with arginines in this mutant (Bylebyl et al, 2003). These results suggest that Red1 prefer- The EMBO Journal VOL 29 | NO 3 | 2010 587