Certainly, further studies are necessary to elucidate the role of ERH in the eukaryotic cell. In this context, the finding of its homologs in fungi, especially those intensively studied, could be very helpful in achieving this objective. Recent progress in the sequencing of various fungal genomes has revealed that some of them indeed possess an ERH homolog, e.g., Mucor circinelloides (genome.jgi-psf.org/Mucci2), Batrachochytrium dendrobatidis (geno- me.jgi-psf.org/Batde5) or Schizosaccharomyces japonicus (for all fission yeast genome sequences see the Internet addresses listed in Materials and Methods). None of those genes has been studied in detail yet but the identification of the ERH homolog in S. japonicus was especially intriguing, since the initial report on the genome of another fission yeast, S. pombe, a closely related and widely used model organism for genetic and cell cycle studies, did not reveal the presence of this gene [7]. Prompted by this finding we performed an independent examination of the S. pombe genome and identified a bona fide homolog of ERH. Not surprisingly, ERH was also missed in various ‘ome-wide analyses performed so far by the fission yeast community [17–20]. Therefore, to characterize for the first time a fungal homolog of the ERH gene, we cloned the ERH genes from all four species of the genus Schizosaccharomyces known to date, S. pombe, S. octosporus, S. cryophilus and S. japonicus, and investigated the role of ERH in S. pombe by studying gene expression and protein localization in wild-type cells, performing functional analysis using gene disrup- tion mutants, and conducting cross-species experiments, also with human ERH. Results The initial report on the S. pombe genome sequence by the Sanger Institute identified 4,824 protein-encoding genes of which none was a homolog of ERH [7]. However, the genomes of two other members of the genus Schizosacccharomyces, S. japonicus and S. octosporus were also sequenced as part of the Fungal Genome Initiative at the Broad Institute and their analysis revealed the presence of ERH-related sequences in both these species as loci SJAG_01867.4 and SOCG_03029.5, respectively [21]. When we evaluated these sequences we found that while the open reading frame (ORF) of the S. japonicus homolog was assembled correctly, the ORF of the S. octosporus gene did not seem to be complete (lacked one exon). In the light of the fact that these two species of the genus Schizosaccharomyces possess an ERH homolog we examined the genome of the third species of the genus, S. pombe, for the presence of an ERH homolog that could be overlooked during the initial automatic assembly of its ORFeome. Not surprisingly, after a BLAST search with the S. japonicus ERH homolog amino acid sequence as a query we identified a bona fide ERH homolog in the S. pombe genome (nucleotides 4066258–4066971 on chromosome 1). An update on the annotation of the S. pombe genome also revealed the presence of this gene as locus SPAC19G12.17 but initially its ORF did not seem to be assembled correctly in the S. pombe genome project database at the Sanger Institute either [22]. Finally, an ERH homolog was identified as locus SPOG_02632.3 in the genome of the fourth, recently discovered species from this genus, S. cryophilus, that was published jointly by the Stowers Institute and the Broad Institute [21,23]. Thus, it became evident that all four currently known species of the genus Schizosaccharomyces possess an ERH gene. ERH Genes from Fission Yeasts Cloning and Sequence Analysis of the ERH Genes and ERH Proteins from All Species of the Genus Schizosaccharomyces To characterize for the first time an ERH homolog from Fungi and to obtain correctly assembled ORFs, we cloned genomic copies and cDNA sequences of the ERH homologs from all four Schizosaccharomyces species, S. pombe, S. octosporus, S. cryophilus and S. japonicus (Figure 1A, B). All the nucleotide sequences of the cloned genomic copies were identical to those available from the Sanger Institute or the Broad Institute. The S. pombe ERH gene (Sperh1+) has a coding sequence (CDS) of 315 bp and codes for a protein of 104 aa (SpErh1p). It is interrupted by five short introns, so the total length from the start to the stop codon, inclusively, in the genomic sequence is 714 bp. The S. octosporus ERH gene (Soerh1+) has a CDS of 315 bp and codes for a 104-aa protein (SoErh1p). It is also interrupted by five introns and its total length is 686 bp. Similary, the S. cryophilus ERH gene (Scerh1+) CDS is 315 bp in length, codes for a 104-aa protein (ScErh1p) and is interrupted by five introns. Its total length is 696 bp. The S. japonicus ERH gene (Sjerh1+) CDS is 330 bp in length and codes for a 109-aa protein (SjErh1p). It is interrupted by six introns and the total length is 780 bp. All these fission yeast ERHs are similar in length to ERHs from other organisms including the 104-aa human ERH, however, the ERH domain (amino acids 3–102 in human ERH) is shortened by four (in SpErh1p, SoErh1p and ScErh1p) or five (in SjErh1p) amino acid residues, which is compensated by additional amino acid residues at the N-terminus of the protein in all four species (Figure 1B) [5,14–16]. Additionally, SjErh1p has extra six amino acids at the C-terminus. The amino acid sequence of SpErh1p displays 28% identity and 54% similarity to human ERH (Figure 1B). SpErh1p has 72% and 71% identities to SoErh1p and ScErh1p, respectively, and only 57% identity to SjErh1p. Overall, only the positions of 50% of the SpErh1p amino acid residues are conserved in all four fission yeast species. Although the SpErh1p primary structure is not highly similar to that of ERHs from other taxa and as a matter of fact the amino acid sequence of ERH is not very well preserved even within the genus Schizosaccharomyces, its secondary and tertiary structures seem to be highly conserved (Figure 1C). The prediction of the Sperh1p three-dimensional structure by the protein structure homology- modeling server SWISS-MODEL using the Protein Data Bank coordinates for human ERH as a template shows a single domain that has all three a helices and four b strands in the same arrangement as found in human ERH, including the long flexible loop between helices a1 and a2 (loop a1-a2, amino acids 46–55 in SpErh1p), which is the region of the weakest similarity among the four fission yeast proteins (Figure 1B) [5,14–16]. The main difference concerns helix a1 which is shortened at the end by four amino acid residues (one full turn) in comparison with human ERH. The lack of another amino acid residue in SjErh1p seems to affect the length of loop a1-a2 in this protein (data not shown). All introns have the GT sequence in the 59 splice site and the AG sequence in the 39 splice site. While the lengths and nucleotide sequences of the introns are rather different in the four Schizosaccharomyces species, their positions have been perfectly conserved during evolution (Figure 1A). Sperh1+, Soerh1+ and Scerh1+ have all their introns in identical positions within their CDSs, as is also true for first four introns of Sjerh1+. The fifth Sjerh1+ intron is closer to the 59 end of the ORF than the fifth introns in the other three genes because the immediately preceding fifth exon is shortened at the 59 end by three nucleotides. This is also the reason for the aforementioned lack of one amino acid residue in the SjErh1p loop a1-a2. Interestingly, there is an AG sequence exactly three nucleotides upstream from the 39 end of the PLOS ONE | www.plosone.org 2 November 2012 | Volume 7 | Issue 11 | e49059