Interactions between MAGE and EID Family Proteins Figure 2. Conserved amino acid residues within the MAGE protein family. Alignment of the N-terminal (A) (aa 89 to 199 of S.p. Nse3) and C- terminal (B) (aa 211 to 301 of S.p. Nse3) part of MHD domain of Nse3/MAGEG1 subfamily. The Nse3/MAGEG1 orthologs are from S. pombe (S.p.), Aspergillus nidulans (A.n.), Neosartorya fischeri (N.f.), Aspergillus terreus (A.t.), Aspergillus clavatus (A.c.), Neurospora crassa (N.c.), Magnaporthe grisea (M.g.), Aspergillus oryzae (A.o.), S. cerevisiae (S.c.), Danio rerio (D.r.), Xenopus tropicalis (X.t.), Galus galus (G.g.), Ornithorhynchus anatinus (O.a.), Monodelphis domestica (M.d.), Dasypus novemcinctus (D.n.), Canis lupus familiaris (C.f.), Mus musculus (M.m.), Homo sapiens (H.s.). Secondary structure derived from the 3D-structure model of Nse3 is indicated above the alignment: cyan rectangle, helix; orange arrow, beta-sheet. Most of the conserved residues were mutated (mut) in the S. pombe Nse3 sequence to alanine; m, mutated residue; red rectangles indicate Nse1- and/or Nse4-specific mutants, respectively; Y264 and L265 residues are labelled with asterisk (B). NSE4b-specific residues of MAGEG1 protein are also indicated in red below the MAGEG1 sequence (B). (C) Alignment of C-terminal part of MHD domain of human MAGE proteins. Shading represents amino acid groups conserved across the family: dark green, hydrophobic and aromatic; light green, polar; blue, acidic; pink, basic; all glycine and proline residues are highlighted in yellow. doi:10.1371/journal.pone.0017270.g002 specifically disrupt binding to Nse4 (Table 1), whilst the Nse3-Nse1 interaction remains undisturbed. Consistent with our pull-down results (Fig. 1B) all these mutations cluster in the C-terminal part of Nse3 (Figure 2B). Based on the sites of these mutations, we deduce that the major part of the Nse4-binding site is formed by hydrophobic residues that are well-conserved in helices H4 (M214, T215, V216, A218, F219, V222, S223), H5 (F235, L236) and H8 (F296, V297, F300). Less well conserved residues from the loop region between helices H5 and H6 (L239, L240, L248, H249) may contribute to the binding as well (Fig. 3B). The hydrophobic pattern of these helices is well conserved within the Nse3/ MAGEG1 subfamily (Figure 2B) as well as across the whole MAGE family (Figure 2C; see below). We conclude that the interaction with Nse4 is mediated by a conserved hydrophobic pocket on the Nse3 surface (Fig. 3B, right panel). Interaction between Nse3 and Nse1 We have identified twelve mutations that specifically destroy the interaction with Nse1 (Table 1). Consistent with the pull-down results (Fig. 1A) most of them cluster within the N-terminal domain of the Nse3 molecule (Figure 2A). Amino acid residues R99, R139 and F147 protrude on the surface, whereas the other residues are partially or fully buried inside the structure of Nse3 (Fig. 3C). We suggest that the latter mutations might disturb the structure of the N-terminal sub-domain of the MHD, while leaving the C-terminal sub-domain containing the Nse4 interaction surface intact. Consistent with our results, human MAGEG1 residues Q94 and F138 (corresponding to R99 and F147 residues in yeast Nse3) contact the NSE1 surface in the 3NW0 co-crystal [14]. We next analysed the effect of Nse1 on the interaction between mutant Nse3 and Nse4 using a yeast-3-hybrid system. In this system, the interaction between Nse3 and Nse4 allowed growth of the cells in the absence of histidine, but not in the presence of 3AT (Figure 4A, row 1). The addition of Nse1 into the system permitted growth in the absence of histidine and in the presence of up to 60 mM 3AT, indicating a much stronger interaction in the presence of Nse1 (Figure 4A, row 2). We analysed the effects of three single mutations in Nse3 that respectively prevented interaction with Nse1 (F147A), Nse4 (F235A) or both (Y264A) in the Y2H system (Fig. 4A, rows 3, 5 and 7) as well as the double mutant Y264A/L265A (row 9). When Nse1 was also present, the interactions between the single mutants of Nse3 and Nse4 (Rows 4, 6 and 8) were indistinguishable from that between wild-type proteins (Row 2). Furthermore the presence of Nse1 partially restored the interaction between the double mutant and Nse4 (Row 10). We conclude that Nse1 markedly strengthens the Nse3- Nse4 interaction (Fig. 1F). Phenotypic analysis of Nse3 mutants We have integrated 19 of the mutations into the genome of S. pombe and analysed the phenotype for temperature-sensitivity as well as for sensitivity to UV light, methyl methanesulfonate (MMS) and HU (Table 1). Because the effects of the mutations on Nse3- Nse4 interaction were largely mitigated by the presence of Nse1 we did not anticipate strong phenotypic effects of the integrated mutations. Indeed, we found that all the single mutants had a normal phenotype, including Y264A and L265A (Figure 4B, rows 3 and 4). Only when we constructed the double mutant Y264A/ L265A was there sensitivity to high temperature, UV irradiation, MMS and HU (Figure 4B, row 2). We conclude from this that the interactions in the context of the whole SMC5-6 complex are considerably stronger than those between two components in isolation. In the former context, mutations that disrupt the two- way interactions are insufficient to cause dissociation within the whole complex. Interactions between MAGEG1 and NSE4b MAGEG1 is the mammalian ortholog of Nse3 and there are two orthologs of Nse4, namely NSE4a and NSE4b [18]. Based on our findings with S. pombe, we have analysed the interactions between a limited number of mutant MAGEG1 (aa 55 to 292) proteins and NSE4b in our yeast-2-hybrid system. Six mutants that change conserved hydrophobic residues in MAGEG1 within its helices H4 (M180, I181, L185) or H8 (F266, V267, V270) (Supplementary Table S2) disrupted this interaction (Figure 5A, row 2). To obtain further support for the disruptive effect of these mutations, we have expressed three of the MAGEG1 mutants (full- length) together with NSE4b in HEK293 cells and examined their interactions with NSE4b by immunoprecipitation from cell extracts (Figure 5B). With two of these mutants the interaction was clearly reduced (lanes 9 and 10) and there was a modest reduction with the third mutant (lane 8). The positions of the mutated amino acids on the structure of MAGEG1 are shown in Figure 5C. These data are in accordance with the S. pombe findings (Figure 2B) and demonstrate the evolutionary conservation of the Nse3-Nse4 binding. MAGEG1-NSE4b effects on transcriptional activation NSE4b/EID3 was first identified as a member of the EID (E1A- like inhibitor of differentiation) family of transcriptional repressors and was shown to inhibit transcriptional activation from several promoters in HuH7 human hepatoma cells [20]. We were interested to see if the interaction between NSE4b and MAGEG1 might affect transcriptional activation, and to examine this, we used the Gal4-SF1 promoter system to study SF-1 mediated transcription activation [20]. Figure 6A confirms that, in HEK293 cells, nuclear receptor stimulates reporter activity some 5–10-fold (columns 1 and 2). Neither NSE4b nor MAGEG1 had much effect (columns 3 and 4), but there was a dramatic concentration- dependent stimulation of transcription activation when MAGEG1 and NSE4b were expressed together at two different concentra- tions of MAGEG1 (columns 8 and 16). To confirm that this transcriptional co-activation was the result of a functional interaction between MAGEG1 and NSE4b, we co-transfected PLoS ONE | www.plosone.org 4 February 2011 | Volume 6 | Issue 2 | e17270