antigen) family of proteins [16,17]. Members of this large protein family have a conserved MAGE-homology domain (MHD). The family is sub-divided into two types. Genes encoding Type I MAGEs (A, B and C sub-families) are expressed only in testis and cancer cells, whereas type II MAGEs are expressed in most tissues. We showed previously that MAGEG1 is the only MAGE protein present in the human SMC5-6 complex and is therefore the ortholog of Nse3 [18]. MAGEG1 has been shown recently to stimulate the E3 ligase activity of human NSE1 [14]. The function of the other MAGE proteins is relatively poorly understood, though there is evidence that several of them are involved with brain development, apoptosis and differentiation [17]. In this paper, we explore the interaction between Nse3 and Nse4 and we identify a conserved hydrophobic pocket on the modelled structure of yeast Nse3 which mediates the interaction with Nse4. We show that the Nse3-Nse4 interaction is conserved in human cells, and that interaction of NSE4b, one of the mammalian orthologs of Nse4, with MAGEG1 results in transcriptional activation in a reporter system. We expand these findings to show that many of the human MAGE proteins are able to react, not only with NSE4a and 4b, but also with related proteins of the EID family. Results Interactions of Nse1, Nse3 and Nse4 In our previous studies, we showed that Nse1, Nse3 and Nse4 (originally Rad62) form a sub-complex of the yeast SMC5-6 octameric complex. We wished to gain a deeper understanding of the detailed interactions within this sub-complex. Previously, we showed that the N-terminal half of Nse1 bound to Nse3 [8]. The MHD of the 328 aa protein Nse3 is comprised of aa 90 to 301. Figure 1A shows that, in pull-down assays, S-tagged fragment (aa 80 to 210) containing the N-terminal part of the MHD is sufficient for binding to in vitro translated full-length Nse1 (lane 6), whereas the C- terminal 107 aa do not bind (lane 9). Conversely the C-terminal fragment of Nse3 binds to Nse4 whereas the N-terminal part does not (Figure 1B, compare lanes 9 and 6). With Nse4, the N-terminal half of Nse4 is sufficient for binding to the C-terminal part of Nse3 (Figure 1C, lane 3) and the C-terminal part of Nse1 (Figure 1D, lane 3), whereas we showed previously that the C-terminal half of Nse4 binds to Smc5 [13]. Good interaction could also be obtained between Nse4 (aa 51 to 260) and Nse1. Using the latter Nse4 construct, we showed that deletion of the RING finger (located between aa 180 and 232) from Nse1 reduced the interaction with Nse4 (Figure 1E, lane 6), as also found by Pebernard et al [19]. These interactions are shown pictorially in Fig. 1F. Interaction of Nse3 and Nse4 To gain further insight into the interaction surfaces, we have mutated most of the conserved residues in the S. pombe Nse3 MHD region (aa 93 to 301, Supplementary Table S1, Figures 2A and B). Each Nse3 mutant was tested for its ability to interact with both Nse1 and Nse4 using the yeast two-hybrid system. 37 out of a total of 82 mutants exhibited no defect in binding to either Nse1 or Nse4 (Table 1). In contrast, 13 mutants lost the ability to interact with both partners. The other 32 mutants disrupted interaction with either Nse1 or Nse4. In order to interpret these data we modelled the structure of Nse3 on the structures of the MHD of MAGEA4 (PDB entry 2WA0) and the recently published structure of MAGEG1 (PDB entry 3NW0 [14]). The structure (Figure 3) is comprised of two domains of approximately equal size, the N- terminal domain being made up of three alpha helices (H1 to H3) and two beta sheets (S1 and S2), whereas the C-terminal domain comprises five helices (H4-8) and two beta sheets (S3 and S4). Interactions between MAGE and EID Family Proteins Figure 1. Interactions between Nse1, Nse3 and Nse4. The indicated His-S-tagged fragments of Nse3 (A, B, C) or Nse1 (D, E) were bound to S-protein agarose-beads and then incubated with in vitro translated Nse1 (A) or Nse4 (B–E). The reaction mixtures were analysed by SDS–12% PAGE gel electrophoresis. The amount of His-S-tagged protein was analysed by immunoblotting with anti-His antibody and the in vitro translated proteins were measured by autoradiography. I, input (5% of total); U, unbound (5%); B, bound (40%). Control, no His-S- tagged protein present. (F) Cartoon of interactions based on panels A–E and our previous work [13]. doi:10.1371/journal.pone.0017270.g001 Most of the 13 mutations that destroy interactions with both Nse1 and Nse4 change residues that are buried inside the Nse3 molecule (Fig. 3A). These amino acid residues most likely maintain the tertiary structure of the MHD. A group of 20 Nse3 mutants PLoS ONE | www.plosone.org 2 February 2011 | Volume 6 | Issue 2 | e17270