Red1 binds Smt3 chains F-M Lin et al Figure 5 Smt3 chains exist in wild-type meiotic cells. Three differ- ent strains expressing V5-Smt3, V5-Smt3-allR or His6–Myc–Smt3 protein are indicated as 1, 2 and 3, respectively. Meiotic cells were harvested at the 5-h time point, and the proteins were trichloroa- cetic acid (TCA) precipitated and dissolved in a denaturing buffer containing 8 M urea. The His6-tagged polypeptides were puriﬁed using Ni2 þ resins. Western blot analysis of total cell lysates (TCLs) and Ni2 þ resin eluants (NREs) was carried out using anti-V5 and anti-Myc antibodies. The molecular weights (in kDa) are indicated to the left of the blots. proteins (Pringle, 1975). Vacuolar proteases (e.g., Pep4, Prb1 and Prc1) are extremely troublesome for the biochemistry of yeast meiosis, because these proteases are not only highly induced upon starvation, but are also essential for sporula- tion (Zubenko and Jones, 1981). Nevertheless, we carried out additional yeast two-hybrid assays to conﬁrm that the Red1– Zip1 interaction is mediated through Smt3 chains. The C-terminal globular domain (residues 849–875) of Zip1 (Zip1C) interacts with Red1C. This interaction was diminished when the same two-hybrid assay was carried out in smt3-allR reporter cells or when Zip1C was replaced by Zip1C3N 3R (Figure 6A). Zip1C3N 3R is incompetent in Smt3 binding because it has three point mutations (E862R, D863R and Q864R) in the SIM (residues 853–864) (Cheng et al, 2006). These results suggest that Smt3 chains are sandwiched between Zip1 and Red1 to promote SC assembly. This hy- pothesis is further supported by an additional line of evi- dence. First, Zip1 and Red1 are both required for the accumulation of Smt3 conjugates or chains in the presence of ZIP3 gene as shown by western blot analysis of the V5-Smt3 expression patterns in wild-type, zip1D and red1D meiotic cells (Figure 6B) and the His6-Myc-Smt3 expression patterns in V5-red1I758R and V5-red13R meiotic cells (Supple- mentary Figure S7). These results are consistent with our earlier report that, as a Smt3 binding protein, Zip1 protects Smt3 chains in the zip3D mutant through competition with Ulp2 (Cheng et al, 2006) (Figure 4B). Second, immunocyto- logical staining of meiotic nuclear spreads indicated that V5-RED1 cells formed complete SCs at the pachytene stage; their chromosomes had strong Zip1 (Figure 6C) and His6–Myc–Smt3 (Figure 6D) staining signals along the entire length. In contrast, V5-red1I758R, V5-red13R and red1D mutants failed to form complete SCs. These three mutants 592 The EMBO Journal VOL 29 | NO 3 | 2010 formed only Zip1 foci or few incomplete SCs. Eventually, Zip1 proteins accumulated into one or a few PCs at later meiotic time points because Smt3 chains could not be stably recruited to the meiotic chromosomes in these mutants (Figure 6E). The order of overall intensity of His6–Myc– Smt3 staining intensity along meiotic chromosomes was V5-RED14V5-red1I758R4V5-red13RBred1D (Figure 6D). Taken together, our results suggest that a mature SC may be established by Smt3 chain bridging AE structural proteins (Red1) and CE structural proteins (Zip1). Discussion Red1, as a binding protein for the Smt3 chain, may have dual functions in the coordination of meiotic recombination and chromosome synapsis. The ﬁrst function promotes Mec1- and Tel1-dependent Hop1 phosphorylation, repressing IS-recom- bination to ensure IH-recombination (Carballo et al, 2008). The second function may be required for recruiting Zip1 and Zip3 proteins to meiotic chromosomes for the initiation of SCs. We suggest that these Zip1–Smt3 chain–Red1 complexes then act together with phosphorylated Hop1 and other ZMM proteins (e.g., Zip2) to promote SC elongation. The use of Smt3 chains for SC assembly is consistent with our previous report that a smt3-allR diploid strain was defective in SC assembly (Cheng et al, 2006), and it explains why the zip3D mutant forms short SCs in the absence of the ‘Zip3-dependent’ Smt3-modiﬁed AE proteins (Agarwal and Roeder, 2000; Hooker and Roeder, 2006). In the latter case, a few Smt3 chains may escape Ulp2 and be sandwiched by Red1 and Zip1 to assemble short SCs. Because Smt3 chains are diffusible and Zip3 has a SIM (Cheng et al, 2006), we propose a ‘relay’ mechanism for SC polymerisation. Zip3 proteins, as well as Ubc9, might be recruited ﬁrst to a CO-designated site (Agarwal and Roeder, 2000; Henderson and Keeney, 2005; Hooker and Roeder, 2006) to promote the formation of Smt3 chains or conjugates. The neighbouring Red1 proteins at the AEs catch these Smt3 chains or con- jugates to recruit not only Zip1 and other ZMM proteins for SC initiation and new Zip3 and Ubc9 proteins to produce more Smt3 chains for SC elongation. The ‘Smt3 chain relay’ hypothesis described here needs to explain how Smt3 chains produced at the site of DSB sites in one chromatid can spread over that chromatid and its homo- logous chromosome without interfering with SC assembly in non-homologous chromosomes. This dilemma can be ad- dressed in three ways. First, the interaction between Smt3 and the SIMs of Red1, Zip1 and Zip3 could be enhanced by Smt3 polymerisation, simply by increasing the local Smt3 concentration, which would facilitate rebinding after disso- ciation. Moreover, longer Smt3 chains diffuse more slowly than shorter Smt3 chains or free Smt3 monomers because of their greater size. Second, Ulp2 can hydrolyse the ‘free’ Smt3 chains. As a result of this hypothesis, Smt3 chains produced in one chromatid are less likely to migrate far enough to reach non-homologous chromosomes. Third, an active sorting me- chanism was recently described for preventing incorrect synapsis between non-homologous chromosomes. This sort- ing and pairing mechanism is accompanied by telomere-led rapid movements (Conrad et al, 2008; Koszul et al, 2008). In principle, the chance of Smt3 chains reaching non-homolo- & 2010 European Molecular Biology Organization