Interactions between MAGE and EID Family Proteins Figure 4. Effect of Nse1 on the interaction between S.pombe Nse3 and Nse4. (A) Yeast-2-hybrid plasmids expressing Nse3, either wild-type or mutated as indicated, fused to the Gal4 DNA-binding domain, and wild-type Nse4 fused to the Gal4 activation domain, were co-expressed with either empty vector (v) or Nse1 (1) in yeast cells, which were subsequently plated in the indicated media and grown at 30uC. AT, 3-aminotriazole. (B) Spot tests of Nse3 wild-type cells (wt:YL), Y264A/L265A (AA), L265A (YA) and Y264A (AL) plated under the indicated conditions. doi:10.1371/journal.pone.0017270.g004 Figure 5. Human MAGEG1 binds NSE4b through conserved hydrophobic surface. (A) Yeast-2-hybrid analysis of the interaction between the indicated mutants of MAGEG1 (aa 55 to 292) and NSE4b (aa 1 to 333) or NSE1 (aa 1 to 266). Interactions result in growth on - Leu,-Trp, -His plates +2 mM AT. Control, no MAGEG1. (B) Co- immunoprecipitation from HEK293 cells co-transfected with S-tagged wild-type and/or mutant MAGEG1 and with FLAG-tagged NSE4b. Lysates were immunoprecipitated with protein-S and immunoblotted with either S-HRP (top) or anti-FLAG (bottom). (C) Structure of the C- terminal domain of MAGEG1 (aa 175 to 270) [14] with the NSE4b- interacting residues indicated in red. doi:10.1371/journal.pone.0017270.g005 a pocket in which the N-terminus of Nse4/NSE4a/4b is located, as shown schematically in Figure 1F and 9B. We have expanded our findings into mammalian systems. We showed previously that there were two NSE4 paralogs in mammals [18]. Using yeast 2-hybrid analysis and co-immunopre- cipitation, we have demonstrated that mutations in MAGEG1 corresponding to those that reduced the interaction with Nse4 in S. pombe, also reduced the interaction of MAGEG1 with NSE4b. To gain further insights into the functional significance of the NSE4b/ MAGEG1 interaction, we used a transcription activation reporter system. Intriguingly, there was a synergistic interaction on transcription activation between MAGEG1 and NSE4b (though not between MAGEG1 and NSE4a – unpublished data), and it was reduced in MAGEG1 mutants that diminished the interaction PLoS ONE | www.plosone.org 8 February 2011 | Volume 6 | Issue 2 | e17270