Red1 binds Smt3 chains F-M Lin et al Figure 1 Red1 preferentially binds Smt3 chains. (A) Quantitative two-hybrid assays were carried out using mitotic reporter host cells carrying either SMT3 or mutant smt3-allR gene. One unit of b-galactosidase hydrolyses 1 mmol of o-nitrophenyl b-D-galactopyr- anoside per min per OD600 unit. Red1C represents the C-terminal fragment (residues 611–827) of the wild-type Red1 protein. The Ile758 residue of SIM-2 was converted to Arg in the Red1CI758R mutant; Red1C2R has two point mutations (Ser715 to Arg and Thr717 to Arg) at the SIM-1. The Red1C3R is the synthetic mutant of the Red1CI758R and Red1C2R mutants. (B) Puriﬁed GST–His6, GST–Red1–His6, GST–Red1I758R–His6, and His6–Myc–Smt3 chains were separated by SDS–PAGE and visualised by Coomassie blue staining. (C) Puriﬁed GST–His6, GST–Red1–His6 and GST– Red1I758R–His6 fusion proteins were ﬁrst bound to GST resins and then incubated with puriﬁed His6–Myc–Smt3 polymeric chains. After extensive washing, the proteins bound to the GST resins were visualised by western blot using anti-Myc antibody. entially associates with Smt3 chains but not with Smt3 monomer or other Smt3 conjugates. To examine if the Red1–Smt3 chain interaction is essential for meiosis, we constructed ﬁve yeast strains expressing V5-tagged Red1 (V5-Red1), V5-Red12R, V5-Red1I758R, V5-Red13R or no Red1 protein. Tetrad analyses revealed that the V5-RED12R mutant, like V5-RED1, generated many viable spores (497%), whereas the V5-red1I758R, V5-red13R and red1D mutants yielded almost no viable spores (o1%). We conclude that SIM-2 not only exhibits a stronger interaction with Smt3 chains than SIM-1, but it is also functionally more important in vivo. 588 The EMBO Journal VOL 29 | NO 3 | 2010 Finally, we examined the afﬁnity of Red1 variant pro- teins for Smt3 chains. We expressed full-length Red1 fusion proteins GST–Red1–His6 and GST–Red1I758R–His6 in Escherichia coli, and then sequentially puriﬁed them on Ni2 þ and glutathione resins. Smt3 chains were synthesised in vitro and partially puriﬁed by size-exclusion gel ﬁltration (Figure 1B). The order of afﬁnity of these proteins had for Smt3 chains was GST–Red1–His6bGST–Red1I758R–His6 B GST–His6 (Figure 1C). Red1CI758R and Red1C3R were also partly defective in homo-oligomerization in the two-hybrid assays carried out in SMT3 and smt3-allR reporter cells, respectively (Supple- mentary Table S1). Although the results obtained from the smt3-allR reporter cells indicate that the Red1–Red1 inter- action is not likely mediated by Smt3 chains, it does not exclude the possibility that other proteins conjugated by Smt3-allR or Smt3 may facilitate the Red1–Red1 interaction. Nevertheless, these two-hybrid results still raise a concern that Red1 homo-oligomerization (Woltering et al, 2000) may be required for the normal functioning of Red1 protein, including the Red1–Smt3 chain interaction. This supposition is not favourable, because the GST–Red1–His6 proteins exhibited much higher afﬁnity to Smt3 chains than the GST–Red1I758R–His6 proteins exhibited (Figure 1C). GST is a dimeric protein on its own, it was used here to induce homo- oligomerization of GST–Red1–His6 and GST–Red1I758R–His6 recombinant proteins. Red1 binds Smt3 chains to promote meiotic IH-recombination The V5-red1I758R and V5-red13R mutant cells had lower stea- dy-state levels of Red1 protein (roughly 70%) compared with wild-type cells (Figure 2A). To ensure that these two mutants did not produce less Red1 proteins than the wild-type V5-RED1 cells, high-copy number vectors expressing V5-Red1I758R or V5-Red13R were then transformed into V5-red1I758R or V5-red13R mutants, respectively (Figure 2B). All of these mutant strains exhibited identical meiotic phe- notypes (i.e., o1% spore viability) before and after being transformed with the supplementary V5-Red1I758R and V5-Red13R expression vectors. Therefore, the meiotic defects of the V5-red1I758R and V5-red13R cells were not because of lower steady-state levels of Red1 proteins. We then carried out cycloheximide shut-off experiments (Penkner et al, 2005) to examine Red1 protein stability. Protein synthesis was inhibited by 200 mg/ml of cyclohexi- mide added into the meiotic cultures at the 4-h time point. Samples were taken at 0, 30, 60, 90, 120 and 180 min after the addition of cycloheximide. Western blot analysis revealed that the levels of V5-Red1, V5-Red1I758R and V5-Red13R remained steady in the presence of cycloheximide upto 180 min (Figure 2D), indicating that the SIM mutation(s) has little or no effect on V5-Red1 protein stability. Immunostaining of sporulating cells and meiotic nuclear spreads with an anti-V5 antibody revealed that V5-Red1I758R and V5-Red13R mutant proteins were properly targeted to nuclei (Supplementary Figure S1) and meiotic chromosomes (Supplementary Figure S2), respectively. We also conﬁrmed that these two mutants were not defective in Hop1 chromo- somal localisation (Supplementary Figure S2) or the Red1– Hop1 interaction (Woltering et al, 2000) (Supplemen- tary Table S2 and Figure S3). & 2010 European Molecular Biology Organization