ERH Genes from Fission Yeasts Figure 4. Effects of erh1+ disruption on S. pombe sensitivity to stresses and on cell cycle progression. (A) Serial dilutions of cells were spotted on YES alone or YES supplemented with 2 M sorbitol, 10 mM hydroxyurea or 0.01% SDS agar plates and incubated for 5 days. Strains used: prototrophic erh1+ ZBM1004 and its erh1D derivative ZBM1005, auxotrophic (ade2 leu 2 ) erh1+ FY7269 and its erh1D derivative ZBM1020, and auxotrophic (ade2 leu2 ura2) erh1+ FY7266 and its erh1D derivative ZBM1030. (B) Cell cycle profiles following nutritional stress in nitrogen-free EMM2 minimal medium. Cells of strains as in (A) were stained with PI and sorted by flow cytometry. Upper series of profiles, cells before nutritional stress (in mid-log phase in YES); lower series of profiles, cells after 48-hour nutritional stress in EMM2-N. Peaks representing 1C and 2C DNA content are indicated. Numbers indicate the percentage of cells with a given DNA content. doi:10.1371/journal.pone.0049059.g004 Chytridiomycota and Zygomycota, often referred to as lower fungi. Here, we show that the ERH homolog is in fact present also in the genus Schizosaccharomyces that belongs to the phylum Ascomycota (Figure 1). However, the fission yeasts seem to be an exception among the higher fungi since, using the fission yeast ERH sequence, we were unable to identify an ERH homolog in the genome sequences of such fungi as S. cerevisiae, A. nidulans or Neurospora crassa, also belonging to Ascomycota, and the button mushroom Agaricus bisporus, the corn smut Ustilago maydis or the stem rust Puccinia graminis, all members of the phylum Basidiomy- cota (M.K. and P.K., unpublished data). The fission yeasts are a unique group of fungi as reflected by their placement into the separate class Schizosaccharomycetes which currently consists of only four species, S. pombe, S. octosporus, S. cryophilus and S. japonicus and diverged from the rest of Ascomycota very early in evolution [21,26–28]. Thus, while the lower fungi and the fission yeasts still possess the ERH gene, it is plausible that most of the higher fungi have lost it during evolution. Conveniently, S. pombe is a fairly simple and extensively studied model organism, so the presence of ERH in its genome can greatly aid the elucidation of the ERH role in the eukaryotic cell, as was the case for numerous other genes and proteins. We cloned the ERH genes from all four species of Schizosacchar- omyces. The proteins coded by these genes show only 25–28% amino acid identity with human ERH, much less than the identity between human ERH and ERH from any other organism (Figure 1B). For example, human ERH displays 52% and 42% identities to the Caenorhabditis elegans and A. thaliana ERHs, respectively [3]. Furthermore, all four fission yeast ERHs show a unique four-amino acid deletion in the middle of the protein. Nevertheless, the secondary and tertiary structures of human ERH are preserved in Erh1p from S. pombe (Figure 1C). The fission yeast ERHs compared to each other display amino acid identities of between 85% (S. cryophilus vs. S. octosporus) and 52% (S. cryophilus vs. S. japonicus); the S. pombe ERH protein shows 72%, 71% and 57% identities to those from S. octosporus, S. cryophilus and S. japonicus, respectively (Figure 1B). Generally, the divergence of ERH sequences between species from a single genus is lower. In Vertebrata, the human, murine and X. laevis ERH proteins are identical; in the genus Drosophila, the ERH proteins of D. melanogaster and D. virilis display 95% identity [3,4]. However, a recent analysis of all four fission yeast genomes has revealed that the average identities between all 1:1 protein orthologs are only 55% for S. pombe and S. japonicus and 85% for S. octosporus and PLOS ONE | www.plosone.org 7 November 2012 | Volume 7 | Issue 11 | e49059