this respect, erh1+ is similar to human ERH which is expressed ubiquitously [9]. To characterize the Erh1p protein we established its intracellular localization in fission yeast. The protein pre- dominantly localized to the nucleus with the exception of the nucleolus, however, a cytoplasmic pool of the protein was seen as well (Figure 2B). This pattern resembles the intracellular localization of human ERH which, however, localizes almost exclusively to the nucleus (Figure 5B) [9]. We also demonstrated that Erh1p exists in two forms of different electrophoretic mobilities, both migrating more slowly than expected (Figure 2C). This probably reflects a posttranslational modification(s) of unknown nature. With the aim of elucidating the ERH function in S. pombe we created strains with disruption of the erh1+ gene in wild-type (prototrophic) and two auxotrophic (ade2 leu2 and ade2 leu2 ura2) genetic backgrounds. First, we looked for abnormalities during normal growth and noticed in the stationary phase in all three erh1D strains accumulation of cells with a comma-like shape, resembling the shape of mutants in ban genes defective in the maintenance of correct direction of cell growth (Figure 3B) [29]. Moreover, when transferred from the stationary phase to minimal medium (EMM2S) all three erh1D strains exhibited a delayed recovery and grew more slowly than did the corresponding erh1+ strains (Figure 3A). That defect was the most profound in the auxotrophic backgrounds. The defect was also found in rich medium (YES), however, it was weak (Figure 3A). When we tested the sensitivity of all three erh1D strains to sorbitol used to induce hyperosmotic stress, hydroxyurea used to inhibit DNA replication and SDS used to destabilize the plasma membrane and, in cells with a defective cell wall, to affect cell integrity, the erh1D disruptants in the auxotrophic backgrounds displayed strong hypersensitivity while the prototrophic erh1D strain showed a less pronounced defect (Figure 4A). Finally, in the auxotrophic erh1D disruptants an increased accumulation of uninucleate cells with a 1C DNA content was observed in response to nutritional stress in nitrogen-free EMM2 minimal medium (Figure 4B). Thus, erh1+ is not essential for vegetative growth in fission yeast, however, Erh1p may play a role in the cellular response to stress conditions. Since hypersensitivity to various stresses was observed it is likely that it could be part of a general stress response. In S. pombe this response seems to be controlled predominantly by a single regulatory system in which key roles are played by the stress-activated MAPK Sty1p (also known as Spc1p or Phh1p) and the transcription factor Atf1p [30,31]. The sty12 mutants are hypersensitive to high osmolarity and hydroxyurea [32,33]. However, in contrast to the erh1D mutant, the sty12 cells are more elongated than wild-type cells [34]. It appears that additional, less prominent systems are also involved in the stress response in fission yeast. Another MAPK, Pmk1p (also known as Spm1p), a constituent of the cell integrity pathway seems to be activated by various stress conditions as well [35]. The pmk1D mutant is hypersensitive to cell wall-damaging agents and displays delayed recovery from the stationary phase, however, it is not hypersensitive to high concentration of sorbitol despite Pmk1p being activated by this agent [35,36]. Moreover, the pmk1D cells are shorter and more rounded [36]. The Tor1p kinase is also required for response to many stress conditions [37,38]. The tor1D cells are hypersensitive to sorbitol [37]. In contrast to the erh1D mutants but similarly to the sty12 cells, the tor1D cells are more elongated [39]. Another TOR protein kinase, Tor2p, is necessary to pass through the G1 phase. However, the tor22 mutations promote arrest in G1 even in rich medium whereas the erh1D mutation facilitates arrest in G1 only on nutritional stress in ERH Genes from Fission Yeasts nitrogen-free minimal medium [40]. In summary, a comparison of the erh1D phenotype with the effects of mutations in genes encoding key constituents of stress response has not indicated clearly in what pathway Erh1p could participate. On the other hand, the observed complex phenotype of the erh1D mutation could be simply viewed as a general diminishing of the fitness of the cells. During normal growth, the phenotype of the erh1D mutation was manifested in both prototrophic and auxotrophic backgrounds but when yeast cells were exposed to stresses, the effects were mainly seen in the auxotrophic backgrounds. Kawai et al. suggested that some growth defects of the tor1D mutation could result from an inability to uptake nutrients efficiently [37]. Others showed that growth of leucine auxotrophs, uracil auxotrophs and, to a lesser extent, histidine auxotrophs but not adenine auxothrophs on minimal medium, that is, in the presence of a high concentration of ammonia, was inhibited by rapamycin, a drug inhibiting TOR kinases, and that the rapamycin-treated tor1+ cells or tor1D cells were defective in leucine uptake, which correlated with reduced expression of three amino acid permeases [41]. We do not think that we observed exactly the same phenomenon with the erh1D mutation in the auxotrophic back- grounds for at least two reasons. Firstly, the erh1D growth defects were also displayed in rich medium which does not contain a high concentration of ammonia (Figure 4A). Secondly, although these defects were also observed in minimal medium in the ade6-M210 leu1-32 background (Figure 5A), adenine was the only supplement added, since the leucine auxotrophy was complemented with the LEU2 gene on the pREP1 plasmid. Interestingly, Tor2p is also involved in regulation of expression of genes coding for amino acid and purine permeases and transporters. In contrast to the tor1D cells, in the tor22 cells numerous such genes were induced [40]. One explanation of the defects observed mainly in the auxotrophic backgrounds could be that supplementation with an auxotrophy- complementing amino acid or nucleobase even at a high concentration does not completely alleviate the auxotrophic defect which, combined with the erh1D mutation, may result in a synthetic growth defect in the presence of some agents. The ERH gene was originally identified in D. melanogaster as a mutation that was manifested only in the r mutant background [1]. The r gene codes for the first three enzymatic activities of the de novo pyrimidine nucleotide biosynthetic pathway and its mutation resulted in pyrimidine deficiency [10]. Our results show that in fact, the effects of the erh1D mutation are fully manifested only in auxotrophic backgrounds, however, both auxotrophic erh1D strains (ade2 leu2 and ade2 leu2 ura2) behaved in the very similar manner. Thus, in S. pombe, pyrimidine auxotrophy is not required for the development of the full erh1D phenotype. The complementation of the erh1D mutation with an exogenous copy of Sperh1+ showed that the observed effects could be attributed solely to that mutation (Figure 5A). The proteins encoded by the Sjerh1+ and human ERH genes display only 57% and 28% identities with SpErh1p, respectively (Figure 1B). These genes complemented the erh1D mutation in a manner that reflected their similarity to Sperh1+ (Figure 5A). The closer homolog rescued the erh1D mutant both in the presence of sorbitol and SDS, while the more distant one only complemented erh1D in the presence of sorbitol. Neither homolog could rescue the erh1D defect in the presence of hydroxyurea. This finding may reflect the interactions of SpErh1p with different partners in response to distinct stresses and, depending on their similarity to SpErh1p, the heterologous ERH proteins could interact with only some of them. No interactor of SpErh1p has been identified yet, however, there are no obvious homologs of the known universal PLOS ONE | www.plosone.org 9 November 2012 | Volume 7 | Issue 11 | e49059