Figure 9. Interactions of MAGE proteins. (A) Data from co- immunoprecipitation experiments were analysed visually; + and 2 signify whether or not an interaction was detected. (B) Cartoon of MAGE interactions showing evolutionary diversification of hypothetical MAGE complexes. doi:10.1371/journal.pone.0017270.g009 1/10th volume of 1 M Tris-HCl pH 8.8. Following reduction and alkylation of samples, proteins were digested with 2.5 ng/ml trypsin for 16 h at 37uC. The resulting peptide mixture was References 1. Lehmann AR, Walicka M, Griffiths DJF, Murray JM, Watts FZ, et al. (1995) The rad18 gene of Schizosaccharomyces pombe defines a new subgroup of the SMC superfamily involved in DNA repair. Mol Cellular Biol 15: 7067–7080. 2. De Piccoli G, Cortes-Ledesma F, Ira G, Torres-Rosell J, Uhle S, et al. (2006) Smc5-Smc6 mediate DNA double-strand-break repair by promoting sister- chromatid recombination. Nat Cell Biol 8: 1032–1034. 3. Murray JM, Carr AM (2007) Smc5/6: a link between DNA repair and unidirectional replication? Nat Rev Mol Cell Biol 9: 177–182. 4. Sollier J, Driscoll R, Castellucci F, Foiani M, Jackson SP, et al. (2009) The Saccharomyces cerevisiae Esc2 and Smc5-6 proteins promote sister chromatid junction-mediated intra-S repair. Mol Biol Cell 20: 1671–1682. 5. Irmisch A, Ampatzidou E, Mizuno K, O’Connell MJ, Murray JM (2009) Smc5/ 6 maintains stalled replication forks in a recombination-competent conforma- tion. EMBO J 28: 144–155. 6. Potts PR, Porteus MH, Yu H (2006) Human SMC5/6 complex promotes sister chromatid homologous recombination by recruiting the SMC1/3 cohesin complex to double-strand breaks. EMBO J 25: 3377–3388. 7. Potts PR, Yu H (2007) The SMC5/6 complex maintains telomere length in ALT cancer cells through SUMOylation of telomere-binding proteins. Nat Struct Mol Biol 14: 581–590. Interactions between MAGE and EID Family Proteins diluted in 0.1% trifluoroacetic acid for analysis by nano-liquid chromatography-tandem mass spectrometry at the Sussex Centre for Proteomics using an LTQ-Orbitrap FT-MS (Thermo Fisher). Tandem mass spectra were extracted by Bioworks version v.3.3 (Thermo Fisher Scientific), and all MS/MS samples were analyzed using Sequest (Thermo Fisher Scientific version SRF v. 5) which was set up to search the ipi.MOUSE.v.3.55 database (55956 entries) with a fragment ion mass tolerance of 1.0 Da and a parent ion tolerance of 5.0 ppm. Deamidation of asparagine, oxidation of methionine and iodoacetamide derivative of cysteine were specified as variable modifications. Scaffold v.3.00.03 (Proteome Software Inc.) was subsequently used to validate the MS/MS based identifications. Peptide identifications were accepted if they could be established at greater than 95.0% probability as specified by the Peptide Prophet algorithm [34], and protein identifications were accepted if they could be established at greater than 99.0% probability and contained at least 2 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm [35]. Supporting Information Table S1 Primers used for site-directed mutagenesis of S. pombe Nse3. (DOC) Table S2 Primers used for site-directed mutagenesis of human MAGEG1. (DOC) Primers used for pTriEx4 constructs. Table S3 (DOC) Acknowledgments We are grateful to Lucas Bowler for the mass spectrometry analysis, to Elaine Taylor for anti-NSE4b antibody and to Eckardt Treuter, Karolinska Inst., Stockholm, for the plasmids used in the transcription activation assays. Author Contributions Conceived and designed the experiments: JJRH KB LK CL MG RC ARL JP. Performed the experiments: JJRH KB LK CL MG RC SV JM JP. Analyzed the data: JJRH KB LK CL MG RC SV JM SRB ARL JP. Contributed reagents/materials/analysis tools: JJRH KB LK CL MG RC ARL JP. Wrote the manuscript: ARL JP. 8. Sergeant J, Taylor E, Palecek J, Fousteri M, Andrews E, et al. (2005) Composition and architecture of the Schizosaccharomyces pombe Rad18 (Smc5-6) complex. Mol Cell Biol 25: 172–184. 9. Zhao X, Blobel G (2005) A SUMO ligase is part of a nuclear multiprotein complex that affects DNA repair and chromosomal organization. Proc Natl Acad Sci U S A 102: 4777–47782. 10. Pebernard S, Wohlschlegel J, McDonald WH, Yates JR, 3rd, Boddy MN (2006) The Nse5-Nse6 dimer mediates DNA repair roles of the Smc5-Smc6 complex. Mol Cell Biol 26: 1617–1630. 11. Andrews EA, Palecek J, Sergeant J, Taylor E, Lehmann AR, et al. (2005) Nse2, a component of the Smc5-6 complex, is a SUMO ligase required for the response to DNA damage. Mol Cell Biol 25: 185–196. 12. Duan X, Sarangi P, Liu X, Rangi GK, Zhao X, et al. (2009) Structural and functional insights into the roles of the Mms21 subunit of the Smc5/6 complex. Mol Cell 35: 657–668. 13. Palecek J, Vidot S, Feng M, Doherty AJ, Lehmann AR (2006) The SMC5-6 DNA repair complex: Bridging of the SMC5-6 heads by the Kleisin, NSE4, and non-Kleisin subunits. J Biol Chem 281: 36952–36959. 14. Doyle JM, Gao J, Wang J, Yang M, Potts PR (2010) MAGE-RING protein complexes comprise a family of E3 ubiquitin ligases. Mol Cell 39: 963–974. PLoS ONE | www.plosone.org 13 February 2011 | Volume 6 | Issue 2 | e17270