ERH Genes from Fission Yeasts Figure 1. ERH genes and ERH proteins from four Schizosaccharomyces species. (A) Intron-exon organization of ERH genes from S. pombe (Sperh1+), S. octosporus (Soerh1+), S. cryophilus (Scerh1+) and S. japonicus (Sjerh1+). Number in parentheses gives total length of CDS plus introns. Black blocks represent exons and are drawn to scale; numbers on top give their lengths in bp. Incisions with numbers indicate intron positions and their length in bp. Consecutive introns are labeled with Roman numerals. (B) Alignment of ERH amino acid sequences from S. pombe (SpErh1p), S. octosporus (SoErh1p), S. cryophilus (ScErh1p) and S. japonicus (SjErh1p). Human ERH is shown as a reference sequence. Numbering according to SpErh1p. Number in parentheses indicates the length of the protein. Dots indicate identical residues and blanks denote missing amino acids. Table shows percent identity of sequences. (C) Predicted three-dimensional structure of Sperh1p generated by SWISS-MODEL using coordinates for human ERH from Protein Data Bank (PDB identifier: 2nmlA). Protein images produced with UCSF Chimera. Helices a1 and a2 and loop a1-a2 in both proteins and the first (Q46) and last (D55) amino acid residues of loop a1-a2 in SpErh1p are indicated. (D) Intron IV-exon V junctions in Sperh1+ and Sjerh1+ and intron I-exon II junction in Sperh1+. Sequences of introns are italicized. The AG sequence of the 39 splice site is underlined and the neighboring AG is denoted by lower case. For details see text. doi:10.1371/journal.pone.0049059.g001 fourth intron of Sjerh1+ (Figure 1D). So it seems likely that the 39 end of this intron ‘‘migrated’’ by three nucleotides into the 59 end of the fifth exon. The last intron of Sjerh1+ does not have a counterpart in any of the other genes and the interruption of the sequence coding for the poorly conserved region at the C-terminus of ERH by this intron is accompanied by the 6-amino acid extension. The introns in all four genes show a significant 59 bias. Four out of five or six introns lie in the first half of the ERH CDS. During cloning of the Sperh1+ cDNA we also isolated two incorrectly spliced sequences (Figure 1D). One of them resulted from the excision of the fourth intron using the AG sequence lying three nucleotides downstream from its correct 39 splice site and coded for a 103-aa polypeptide that lacked one amino acid residue (alanine) in loop a1-a2. In this respect it resembled the splicing pattern of Sjerh1+. The other cDNA resulted from the excision of the first intron together with most of the second exon due to using the AG sequence located 20 nucleotides downstream from its correct 39 splice site and coded for a 28-aa oligopeptide only because of the reading frame shift and creation of a premature stop codon. During cloning of the Scerh1+ cDNA we isolated one sequence that lacked the whole fourth exon presumably because of the co-excision of the third and fourth introns. We did not find any incorrectly spliced sequences during cloning of the cDNAs of the two other genes. Initial Characterization of the erh1+ Gene and Erh1p Protein from S. pombe To establish the expression pattern of erh1+ during vegetative growth and adaptation to unfavorable conditions in S. pombe we isolated total RNA from the major stages and performed Northern analysis using radiolabeled erh1+ cDNA as a probe (Figure 2A). The erh1+ transcript was present at a similar level in all stages examined: both the haploid and diploid cultured to the mid-log phase, the haploid cultured to the stationary phase, both the haploid and diploid subjected to nutritional stress in nitrogen-free EMM2 (EMM2-N) minimal medium, and the haploid subjected to nutritional stress in low-glucose EMM2 minimal medium or to hyperosmotic stress. Erh1p was not included in proteome-wide studies on the intracellular localization of S. pombe proteins [19,20]. Our analysis of its sequence did not reveal any targeting signal. Thus, to determine the intracellular localization of Erh1p we introduced plasmid pREP1/SpErh1p-link-yEGFP coding for Erh1p tagged on the C-terminus with yEGFP (yeast-enhanced green fluores- cence protein) into strain FY7269 and analyzed the resulting cells (strain ZBM1021) by fluorescence microscopy (Figure 2B). Erh1p- yEGFP was clearly present in the nucleus, with the exception of the nucleolus. However, there was also a significant amount of Erh1p-yEGFP in the cytoplasm. To exclude the possibility that the presence of Erh1p-yEGFP in the cytoplasm resulted from its overexpression (pREP1 is a high-copy number expression plasmid with a strong promoter, nmt1), we chromosomally tagged erh1+ with yEGFP at its 39 end in the same strain (FY7269). In the resulting cells (strain ZBM1028) the expression of Erh1p-yEGFP under the control of its own promoter was weaker but the protein was still observed in the cytoplasm (Figure 2B). To further characterize Erh1p we introduced plasmid pREP1/ SpErh1p-3HA coding for Erh1p tagged on the C-terminus with 3HA (a triple hemagglutinin epitope) of the theoretical molecular weight of 16907 Da into strain FY7269. Transformant (strain ZBM1022) cells were lysed and after immunoprecipitation with an anti-HA antibody precipitates were separated by SDS/poly- acrylamide gel electrophoresis and stained with silver (Figure 2C). Two strong protein bands with electrophoretic mobilities of approx. 22 and 23 kDa were consistently detected. These bands were absent in the precipitate obtained from cells of the control strain ZBM1023 (strain FY7269 transformed with plasmid pREP1). Tandem mass spectrometry analysis of these two bands unveiled the presence of numerous tryptic peptides corresponding to Erh1p-3HA in both of them (79% and 67% of protein sequence coverage for the lower and upper band, respectively), indicating that they indeed were two forms of this protein (data not shown). Functional Analysis of the erh1+ Gene from S. pombe To investigate the role of Erh1p in S. pombe we disrupted erh1+ through replacement of the full coding region with a kanamycin cassette by double homologous recombination in the prototrophic strain ZBM1004 and auxotrophic strains FY7269 (ade2 leu2) and FY7266 (ade2 leu2 ura2). The obtained disruptants (erh1D strains), ZBM1005, ZBM1020 and ZBM1030, respectively, were tested for growth defects in various conditions. When the strains were cultured in EMM2S minimal medium after transferring from the stationary phase all three disruptants displayed a delay in recovery from the stationary phase, grew more slowly and reached lower final optical densities (OD600) than did the corresponding erh1+ strains (Figure 3A). The auxotrophic erh1D strains, especially ZBM1030 (ade2 leu2 ura2), exhibited a stronger growth defect than the prototrophic erh1D strain. When the strains were cultured in YES rich medium after transferring from the stationary phase the defect was only minimal for all three disruptants (Figure 3A). The morphology of the erh1D disruptant cells was examined by light microscopy with Hoffman Modulation Contrast. Cells with a comma-like shape accumulated in cultures of all three disruptants when they reached the stationary phase in YES (Figure 3B) or EMM2S (data not shown). To find other possible phenotypes associated with the erh1D mutation we studied the sensitivity of the erh1D strains to the following stress conditions: hyperosmotic stress (sorbitol), in- hibition of DNA replication (hydroxyurea) and destabilization of the plasma membrane (SDS) using a serial dilution method (Figure 4A). Both auxotrophic erh1D strains showed hypersensi- tivity to all these agents. The prototrophic erh1D strain exhibited hypersensitivity to sorbitol similar to the auxotrophic erh1D strains. PLOS ONE | www.plosone.org 4 November 2012 | Volume 7 | Issue 11 | e49059