ERH Genes from Fission Yeasts Figure 2. Expression of erh1+ and characterization of Erh1p in S. pombe. (A) Northern analysis of erh1+ transcript. Total RNA from haploid strain FY12697 cultured to mid-log phase in YES, cultured to stationary phase in YES, subjected to nutritional stress in low-glucose EMM2, subjected to nutritional stress in EMM2-N or subjected to hyperosmotic stress in YES supplemented with 2 M sorbitol and a diploid constructed freshly by crossing strains FY12697 and FY7519, cultured to mid-log phase in YES or subjected to nutritional stress in EMM2-N, separated by 1.2% formaldehyde/agarose gel electrophoresis and stained with ethidium bromide (upper panel) followed by transfer to membrane and hybridization with radiolabeled erh1+ cDNA (lower panel). (B) Intracellular localization of Erh1p. Upper series of images, cells expressing yEGFP-tagged Erh1p from pREP1 (strain ZBM1021) visualized with Nomarski Interference Contrast, nuclei stained with DAPI and localization of yEGFP-tagged Erh1p determined by direct fluorescence; lower series of images, cells expressing yEGFP-tagged Erh1p from erh1+ chromosomal locus (strain ZBM1028), nuclei stained with DAPI and localization of yEGFP-tagged Erh1p determined by direct fluorescence. (C) Identification of two forms of Erh1p. Immunoprecipitates with anti-HA monoclonal antibody from lysed cells expressing 3HA-tagged Erh1p (strain ZBM1022) or negative control (strain ZBM1023) separated by 15% SDS/polyacrylamide gel electrophoresis and stained with silver. Protein molecular mass standard of 21.5 kDa is shown to the left. Both protein bands (p22 and p23) were identified by tandem mass spectrometry as Erh1p-3HA. doi:10.1371/journal.pone.0049059.g002 However, it was slightly less hypersensitive to hydroxyurea than the auxotrophic erh1D strains and showed only a minimal defect when grown in the presence of SDS. We next analyzed the progression of the cell cycle in the erh1D strains using flow cytometry. The cell cycle of exponentially growing haploid fission yeast cells displays two unusual features. The G2 phase is very long, taking 70% of the generation time, and the completion of cytokinesis is delayed until the late S phase, which together results in approx. 90% of cells having a 2C DNA content (all G2, M and G1 phase cells) [24,25]. However, following nitrogen deprivation, accumulation of uninucleate cells with a 1C DNA content is observed [24]. Switching from rich medium to nitrogen-free minimal medium leads to nutritional stress which is not only limited to nitrogen deprivation since the latter medium also lacks many organic compounds including amino acids and nucleotides. Thus, such transfer could also disturb the cell cycle. The erh1D strains cultured to the mid-log phase in YES exhibited 90.8–93.1% of cells with a 2C DNA content, the same as in the corresponding erh1+ strains (Figure 4B). When subjected to nutritional stress in nitrogen-free EMM2 minimal medium for 48 hours, all strains showed a profile with an additional 1C peak. However, the proportion of cells with a 1C DNA content was lower in the auxotrophic erh1+ strains than in the prototrophic erh1+ strain (29.3% and 36.6% vs. 43.1% in FY7269, FY7266 and ZBM1004, respectively). Significantly, the auxotrophic erh1D strains exhibited a much higher percentage of 1C cells (52.2% and 60.3% in ZBM1030 and ZBM1020, respectively), showing that they were more readily arrested in the G1 phase in response to the nutritional stress than the corresponding erh1+ strains. The prototrophic erh1D strain showed only a small increase (to 49.5%) in the proportion of 1C cells in comparison with the prototrophic erh1+ strain. Complementation of the erh1D Mutation and Cross- species Experiments To exclude the possibility that the observed growth defects were not due to the erh1D disruption only, we tested the ability of an exogenous copy of Sperh1+ to rescue these defects. We introduced plasmids pREP1 and pREP1/SpErh1p into strain ZBM1020 and the resulting transformants (strains ZBM1024 and ZBM1025, respectively) together with strain ZBM1023 were tested for growth in the presence of the same stress-inducing agents as above (Figure 5A). As expected, ZBM1024 exhibited hypersensitivity to each agent when compared to ZBM1023, and ZBM1025 showed no defect in the presence of hydroxyurea or SDS and even reduced sensitivity to sorbitol. To find whether the functions are conserved despite the relatively low amino acid sequence identities between the ERH orthologs from the different species of the fission yeasts or between any fission yeast species and humans, we determined the ability of the S. japonicus and human ERH genes to complement the erh1D mutation in S. pombe. We introduced plasmids pREP1/SjErh1p and pREP1/HsERH into strain ZBM1020 and the resulting transformants (strains ZBM1026 and ZBM1027, respectively) were tested for growth on the same media as above (Figure 5A). While the Sjerh1+ cDNA did rescue the erh1D growth defect in the presence of sorbitol or SDS, it was rather insufficient in the presence of hydroxyurea. The ERH cDNA rescued the erh1D mutant only in the presence of sorbitol. To further study SpErh1p we performed heterologous expres- sion of Sperh1+. Human HeLa cells were transfected with plasmids PLOS ONE | www.plosone.org 5 November 2012 | Volume 7 | Issue 11 | e49059