interactors of human ERH, Ciz1 and PDIP46/SKAR, in the fission yeast proteome (M.K. and P.K., unpublished data). Due to the moderate degree of identity of SpErh1p with human ERH, yet almost identical secondary and tertiary structure (Figure 1B, C), its localization to the nucleus (Figure 2B) and the fact that ERH complemented at least partially the erh1D mutation (Figure 5A), SpErh1p could be a valuable molecular tool to study the interaction of ERH with its two nuclear partners, Ciz1 and PDIP46/SKAR, e.g., in searching for amino acid residues responsible for the interactions. We found that SpErh1p expressed in HeLa cells did enter the nucleus (Figure 5B), but in contrast to ERH, it was not recruited to the Ciz1-containing replication foci suggesting a lack of an interaction between SpErh1p and Ciz1. No interaction between SpErh1p and PDIP46/SKAR and also between SpErh1p and Ciz1 was found by yeast two-hybrid system (Figure 5C). These results suggest that the amino acid residues shared between ERH and SpErh1p are not sufficient for an interaction with Ciz1 or PDIP46/SKAR and further studies are necessary to identify the ERH residues responsible for these interactions. In conclusion, we posit that the ERH protein was present in the nucleus of the last common ancestor of all eukaryotes in which it played a role in the response to adverse environmental conditions. However, the ERH gene was not essential for normal growth and during evolution of eukaryotes, ancestors of some species, in- cluding most of the higher fungi have lost this gene, possibly because its role could be fulfilled by another protein. In the higher fungi several independent ERH loss events must have occurred, since the divergence of Ascomycota and Basidiomycota obviously preceded the separation of the class Schizosaccharomycetes from the rest of Ascomycota. On the other hand, with an increase of the proteome size and accumulation of changes in the primary structure of ERH, the protein became involved in interactions with some novel acquisitions, i.e., Ciz1 and PDIP46/SKAR in Vertebrata and gained additional functions while the primary one could be restricted or even completely eliminated in some evolutionary lineages. In turn, the complexity of its interactions constrained further evolution of this small, single-domain protein. Materials and Methods Databases and Bioinformatic Analyses For S. pombe the genome sequence at the Sanger Institute (www. sanger.ac.uk/Projects/S_pombe) was used. For S. octosporus, S. cryophilus and S. japonicus the collection of fission yeast genome sequences available at the Broad Institute (www.broadinstitute. org/annotation/genome/schizosaccharomyces_group) was em- ployed. DNA analyses were performed with the BLAST program [42]. Amino acid sequence alignments were performed using the Clustal X 2.0 program and manual adjustment [43]. Protein structures were predicted by the SWISS-MODEL server (swiss- model.expasy.org) using the Protein Data Bank (www.rcsb.org/ pdb) coordinates for the human ERH protein (PDB identifier: 2nmlA) [44]. Protein images were produced with the UCSF Chimera package [45]. General Techniques for the Fission Yeasts The fission yeast strains used in this study are listed in Table 1. Standard methods for S. pombe were used as described [18]. All four species were grown on YES (YE rich medium supplemented with adenine, uracil, leucine, lysine and histidine, each at 225 mg/ l) at 30uC, with the exception of S. cryophilus which was cultivated at 25uC. For S. pombe the following variations of the EMM2 minimal medium were also used: EMM2S (EMM2 supplemented ERH Genes from Fission Yeasts as above), EMM2+ADE (EMM2 supplemented with adenine at 100 mg/l), EMM2-N (nitrogen-free EMM2) and low-glucose EMM2 (EMM2 with a glucose concentration lowered to 0.5%). For growth curve determination two-and-a-half-day YES or EMM2S precultures were diluted to a starting OD600 of 0.1 in YES and EMM2S, respectively, and grown with shaking for up to 72 hours (until the stationary phase was reached). For growth tests cells were cultured to the mid-log phase in YES or EMM2+ADE, washed with saline and ten-fold serial dilutions were spotted on plates with YES or EMM2+ADE agar alone and YES or EMM2+ADE agar supplemented with 2 M sorbitol, 10 mM hydroxyurea or 0.005–0.01% SDS and were grown for 5 days. For nutritional stresses cells were cultured to the mid-log phase in YES, washed three times with saline and transferred to EMM2-N and incubated with shaking for 48 hours or transferred to low-glucose EMM2 and incubated with shaking for 24 hours. For hyper- osmotic stress cells were cultured to the mid-log phase in YES, transferred to YES supplemented with 2 M sorbitol and incubated with shaking for 24 hours. Transformations of strains FY7269 and ZBM1020 with pREP1 or derivatives were done by the lithium acetate method with sonicated salmon sperm DNA as carrier and incubation with DMSO, and transformants were selected on EMM2+ADE agar. For construction of a diploid, strains FY12697 and FY7519 were crossed on ME agar at 25uC and after streaking out on YES agar supplemented with Phloxin B (5 mg/ml) diploid clones (stained dark red) were identified. For construction of the prototrophic haploid ZBM1004, strains FY7269 and FY7519 were crossed as above and the ade+ leu+ meiotic progeny was selected on EMM2 agar followed by determination of the mating type by polymerase chain reaction (PCR) with the common (59-AGAA- GAGAGAGTAGTTGAAG-39), h+ specific (59-ACGGTAGT- CATCGGTCTTCC-39) and h2 specific (59-TACGTTCAGTA- GACGTAGTG-39) primers. Total DNA, RNA and cDNA Total DNA was liberated from cells by heating their suspension in water for 5 minutes at 95uC and removing cellular debris by spinning at 132006g for 3 minutes at room temperature. For total RNA preparation, cells were pulverized in liquid nitrogen and RNA was isolated using TRI Reagent (Sigma) followed by purification with NucleoSpin RNA Clean-up (Macherey-Nagel). For total cDNA preparation, 2 mg of total RNA was used with RevertAid M-MuLV Reverse Transcriptase (Fermentas) accord- ing to the manufacturer’s recommended protocol. Northern Analysis Total RNA was resolved on a 1.2% formaldehyde/agarose gel using RiboRuler High Range RNA Ladder (Fermentas) as RNA size standards and transferred to Hybond-N+ nylon membrane (Amersham) by capillary blotting. The cloned Sperh1+ cDNA was labeled with [a-32P]dATP (Hartmann Analytic) using Megaprime DNA labelling system (Amersham) and high stringency hybrid- ization was carried out overnight at 65uC in the modified Church and Gilbert buffer according to the protocol recommended by Amersham. After washing, the membrane was exposed for one week to an X-ray film with an intensifying screen at 280uC. Immunoprecipitation Yeast cells in the early stationary phase from 2 liters of EMM2+ADE were pelleted, frozen in liquid nitrogen and pulverized manually. The obtained powder was suspended in lysis buffer (30 mM Tris-HCl, 150 mM NaCl, 0.05% NP-40, 1 mM PMSF, pH 8.0), the suspension was cleared by centrifugation (400006g for 60 minutes at 4uC) and the supernatant was dialyzed PLOS ONE | www.plosone.org 10 November 2012 | Volume 7 | Issue 11 | e49059