Interactions between MAGE and EID Family Proteins Figure 3. Interacting residues of Nse3 modelled on the crystal structures of MAGEA4 (PDB 2WA0) and MAGEG1 (PDB 3NW0). Homology modelling was used to generate the predicted S. pombe Nse3 MHD structure. Ribbon representation (left panels) of the predicted Nse3 3D structure model with helices (cyan) and beta-sheets (orange) indicated as in Figure 2. Right panels represent surface views. (A) The residues that, when mutated, lost their ability to interact with both Nse1 and Nse4 interacting partners are buried inside (indicated in dark blue). (B) Sequence and structure of Nse3 (aa 211 to 300) showing which mutations inhibit interaction with Nse4 (red). Top view of the structure shown in panel (A). (C) Residues in the N-terminal domain (aa 92 to 187) that, when mutated, reduce the interaction with Nse1 are indicated in green. The small cartoons at the left of the panels are miniatures of the full-length structure. The parts indicated in red are expanded in the main panels. doi:10.1371/journal.pone.0017270.g003 Evolutionary conservation of interactions between MAGE and EID proteins The MAGE family consists of a single family member (the ortholog of Nse3) in all eukaryotic organisms except for placental mammals. In contrast, in placental mammals, there are tens of MAGE gene (and pseudogene) copies per genome ([16]; JP unpublished data). For example, the human genome contains 22 class I and 11 class II MAGE genes (Table 2 and Figure 2C). However, we showed previously that only MAGEG1 is found in the SMC5-6 complex and is the true ortholog of Nse3 [18]. Of the two orthologs of Nse4, only NSE4a was detected in the SMC5-6 complex from cultured cells, but we showed that, when overexpressed following transfection, either paralog could be incorporated into the complex [18]. Examination of EST libraries suggested that NSE4b was expressed mainly in the testis and tissue-specific micro-array data show that, in the mouse, it is expressed exclusively in testis (http://biogps.gnf.org/#goto= genereport&id=493861). This raised the possibility that it might be the SMC5-6 kleisin in the testis. To test this directly, we used antibodies against NSE4b for immunoprecipitations from extracts of mouse testes. The immunoprecipitates were analysed for other components of the SMC5-6 complex by immunoblotting with anti-hSMC6 and anti-hNSE2/MMS21. Figure 7A, lane 5, shows that both mSMC6 and mNSE2/mMMS21 were co-immunopre- cipitated. Finally we immunoprecipitated SMC6 from mouse testes and analysed the immunoprecipitates by mass spectrometry. NSE4b (as well as NSE4a) and other expected members of the complex were detected (data not shown), confirming that NSE4b is a testis-specific component of SMC5-6. In Figure 7B (lane 6), we confirm that, when expressed in HEK293 cells, immunoprecipitation of MAGEG1 coprecipitates both NSE4b and NSE1 [18]. However the hydrophobic character of the Nse4-interacting residues in Nse3/MAGEG1 is well conserved in the sequences of all the human MAGE proteins (Figure 2C), suggesting that the Nse3-Nse4 interaction might be conserved more widely. We previously showed that FLAG-tagged NSE4b could interact with NSE1 and SMC6, presumably as part of the SMC5-6 complex [18]. The immunoprecipitation shown in Figure 7B (lane 12, bottom panel) confirms the interaction of NSE4b with NSE1, but also shows an interaction with MAGEA1 (lane 12, middle panel). When we did the immunoprecipitation the other way round, immunoprecipitating MAGEA1, the interaction with NSE4b was confirmed (lane 18, top panel), but there was minimal interaction with NSE1 (lane 18, bottom panel), suggesting that the NSE4b-MAGEA1 formed a complex that was separate from the SMC5-6 complex. To extend these findings, we have co- expressed representative S-tagged MAGE proteins with FLAG- tagged NSE4a or b and analysed the interactions by co- immunoprecipitation. The results are shown in Figures 7C–H. Interestingly most of the MAGE proteins tested interacted significantly with both NSE4 paralogs (lanes 3 and 6, lower panels in each figure section). Figure 7C–H show clear interaction of both paralogs with the Type I MAGE A1 (C) and the type II MAGE D4b (D) and necdin (G). These can be compared with the previously described interactions of MAGEG1 with NSE4a and b (Figure 7F and [18]), which are known to be components of the SMC5-6 complex. An exception is MAGEF1, which does not appear to interact with either paralog (Figure 7E). The N-terminal part of yeast Nse4 mediates the interaction with Nse3 (Fig. 1C). Interestingly, NSE4a and NSE4b/EID3 are members of another gene family, the EID family, whose other members, namely EID1, 2 and 2b (Table 2), have substantial sequence identity to the N-terminal part of the Nse4 proteins (Figure 8A and [20]). Interactions of S-tagged MAGE proteins co- expressed with FLAG-tagged EID1, 2 and 2b in HEK293 cells are shown in Figure 8B–G. As with the NSE4 paralogs (Figure 7), we found that the MAGE proteins interacted with the EID proteins, albeit to different extents. Interestingly MAGEG1 did not interact with any of the EID proteins (Figure 8E, lanes 3, 6, 9) while MAGEF1 precipitated all of them (Fig. 8D, lanes 3, 6, 9). Because of different levels of expression of the different MAGE proteins, it is not possible to make quantitative comparisons, but a summary of all the interactions that we have analysed is presented in Figure 9A. Effects of MAGE and EID proteins on gene expression When we analysed the effect of several different MAGE proteins in the transcription activation system, MAGEA1 and MAGED4b had large stimulatory effects on Gal4-SF1 activity in HEK293 cells (Fig. 6B, columns 6 and 10), which were completely abolished by EID1 (columns 7 and 11) but were unaffected by NSE4b (columns 8 and 12). Necdin alone had little transactivation activity (column 14), but, in its presence, the reporter activity was resistant to inhibition by EID1 (column 15), (in keeping with the findings of Bush and Wevrick [21]), and NSE4b also had little effect (column 16). None of the MAGE/EID interactions resulted in transcrip- tional co-activation as found between MAGEG1 and NSE4b (Fig. 6A). Discussion In our previous studies we showed that Nse1, Nse3 and Nse4 formed a sub-complex within the highly conserved SMC5-6 protein complex and that Nse3 was structurally homologous to the MAGE protein family [8]. We have now refined the architectural definition of this sub-complex and focussed on the Nse3/MAGE protein. We have identified a surface on Nse3 that interacts with Nse4 and a structural domain of Nse3 that interacts with Nse1. This analysis is based on modelling the structure of Nse3 onto the structure of MAGEA4 and G1 deposited in the Protein Database. The validity of our conclusions obviously depends on the accuracy of our modelling. The high level of sequence similarity between MAGE proteins and Nse3 together with the internal self- consistency of our observations gives us confidence that our modelling is reasonably accurate. The interacting region between S. pombe Nse1 and Nse3 that we have defined, based on our two- hybrid and modelling analysis, corresponds well with that deduced from the crystal structure of the orthologous human NSE1- MAGEG1 [14]. Furthermore NSE1 and the hydrophobic cleft on Nse3/MAGEG1 that we predict forms the interaction surface with Nse4 are positioned on the same face of Nse3/MAGEG1. We predict that Nse1/NSE1 and the hydrophobic cleft together form PLoS ONE | www.plosone.org 7 February 2011 | Volume 6 | Issue 2 | e17270