ERH Genes from Fission Yeasts Figure 3. Effects of erh1+ disruption in S. pombe on growth rate and cell morphology. (A) Growth curves for three erh1+ strains (solid lines), prototrophic ZBM1004, auxotrophic (ade2 leu2) FY7269 and auxotrophic (ade2 leu2 ura2) FY7266 and three corresponding erh1D strains (dashed lines), ZBM1004 derivative ZBM1005, FY7269 derivative ZBM1020 and FY7266 derivative ZBM1030 in EMM2S minimal medium (left) or in YES rich medium (right). Cultures were inoculated to OD600 of 0.1 from stationary cultures in the same medium. (B) Cell morphology by light microscopy with Hoffman Modulation Contrast. Images of strains as in (A) cultured to stationary phase in YES. doi:10.1371/journal.pone.0049059.g003 pEGFP-N1/SpErh1p and pEGFP-N1/ER coding for SpErh1p and human ERH, respectively, both tagged on their C-termini with EGFP, and were analyzed by fluorescence microscopy after 48 hours (Figure 5B). As expected, ERH localized exclusively to the nucleus [9,11]. In contrast to the predominantly nuclear localization of SpErh1p in yeast cells (Figure 2B), this protein was equally present in the nucleus and cytoplasm of HeLa cells. We next tested the ability of SpErh1p to interact with a universal molecular partner of human ERH, the Ciz1 protein which localizes to DNA replication foci in the nucleus. HeLa cells were cotransfected with plasmids pEGFP-N1/SpErh1p and pmCherry- N1/Ciz1 coding for human Ciz1 tagged on the C-terminus with the red fluorescent protein mCherry and analyzed by fluorescence microscopy (Figure 5B). HeLa cells cotransfected with pEGFP- N1/ER and pmCherry-N1/Ciz1 were used as a positive control. While ERH moved to the Ciz1-containing replication foci as reported previously [11], SpErh1p remained diffused in the nucleoplasm. The possibility of interaction of SpErh1p with another universal molecular partner of human ERH, the PDIP46/SKAR protein, was tested using the yeast two-hybrid system which had been used to identify the both ERH interactors previously [9,11]. The host S. cerevisiae strain L40 was transformed with plasmid pHybLex/Zeo- SpErh1p coding for SpErh1p fused to the C-terminus of the LexA DNA-binding domain. The LexA-SpErh1p chimeric protein alone did not activate the LacZ reporter gene in the bait strain (data not shown). Next, the bait strain was transformed with plasmid pYESTrp2/PDIP46 coding for human PDIP46/SKAR fused to the C-terminus of the B42 transcription activation domain. The bait strain was also transformed with plasmid pYESTrp2/Ciz1 coding for human Ciz1 fused to the C-terminus of B42. SpErh1p failed to interact with PDIP46/SKAR or Ciz1 (Figure 5C). Discussion The ERH gene is present in genomes of most eukaryotic organisms including Animalia (from the sponge Amphimedon queenslandica to the tapeworm Echinococcus multilocularis to humans), Plantae (from the green alga Chlorella sp. to the moss Physcomitrella patens to the flowering plant Arabidopsis thaliana) and Protista (e.g., the protozoan Toxoplasma gondii, the red alga Cyanidioschyzon merolae, the water mold Saprolegnia parasitica or the slime mold Dictyostelium discoideum), which strongly suggests that it was present also in the genome of the last common ancestor of all eukaryotes (M.K. and P.K., unpublished data). In this respect, Fungi are striking exception since this gene has only been identified in the phyla PLOS ONE | www.plosone.org 6 November 2012 | Volume 7 | Issue 11 | e49059