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 α1 and α2 and loop α1-α2 in both proteins and the first (Q46) and last (D55) amino acid residues of loop α1-α2 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 3′ splice site is underlined and the neighboring AG is denoted by lower case. For details see text.
Figure 2.
Expression of erh1+ and characterization of Erh1p in S. pombe.
(A) Northern analysis of erh1+ transcript. Total RNA from haploid strain FY12697 cultured to mid-log phase in YES, cultured to stationary phase in YES, subjected to nutritional stress in low-glucose EMM2, subjected to nutritional stress in EMM2-N or subjected to hyperosmotic stress in YES supplemented with 2 M sorbitol and a diploid constructed freshly by crossing strains FY12697 and FY7519, cultured to mid-log phase in YES or subjected to nutritional stress in EMM2-N, separated by 1.2% formaldehyde/agarose gel electrophoresis and stained with ethidium bromide (upper panel) followed by transfer to membrane and hybridization with radiolabeled erh1+ cDNA (lower panel). (B) Intracellular localization of Erh1p. Upper series of images, cells expressing yEGFP-tagged Erh1p from pREP1 (strain ZBM1021) visualized with Nomarski Interference Contrast, nuclei stained with DAPI and localization of yEGFP-tagged Erh1p determined by direct fluorescence; lower series of images, cells expressing yEGFP-tagged Erh1p from erh1+ chromosomal locus (strain ZBM1028), nuclei stained with DAPI and localization of yEGFP-tagged Erh1p determined by direct fluorescence. (C) Identification of two forms of Erh1p. Immunoprecipitates with anti-HA monoclonal antibody from lysed cells expressing 3HA-tagged Erh1p (strain ZBM1022) or negative control (strain ZBM1023) separated by 15% SDS/polyacrylamide gel electrophoresis and stained with silver. Protein molecular mass standard of 21.5 kDa is shown to the left. Both protein bands (p22 and p23) were identified by tandem mass spectrometry as Erh1p-3HA.
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 (ade− leu−) FY7269 and auxotrophic (ade− leu− ura−) FY7266 and three corresponding erh1Δ 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.
Figure 4.
Effects of erh1+ disruption on S. pombe sensitivity to stresses and on cell cycle progression.
(A) Serial dilutions of cells were spotted on YES alone or YES supplemented with 2 M sorbitol, 10 mM hydroxyurea or 0.01% SDS agar plates and incubated for 5 days. Strains used: prototrophic erh1+ ZBM1004 and its erh1Δ derivative ZBM1005, auxotrophic (ade− leu−) erh1+ FY7269 and its erh1Δ derivative ZBM1020, and auxotrophic (ade− leu− ura−) erh1+ FY7266 and its erh1Δ derivative ZBM1030. (B) Cell cycle profiles following nutritional stress in nitrogen-free EMM2 minimal medium. Cells of strains as in (A) were stained with PI and sorted by flow cytometry. Upper series of profiles, cells before nutritional stress (in mid-log phase in YES); lower series of profiles, cells after 48-hour nutritional stress in EMM2-N. Peaks representing 1C and 2C DNA content are indicated. Numbers indicate the percentage of cells with a given DNA content.
Figure 5.
Complementation of erh1Δ mutation and cross-species experiments.
(A) Serial dilutions of cells were spotted on EMM2+ADE alone or EMM2+ADE supplemented with 2 M sorbitol, 10 mM hydroxyurea or 0.005% SDS agar plates and incubated for 5 days. Strains used: auxotrophic (ade− leu−) erh1+ FY7269 transformed with pREP1 (ZBM1023), FY7269 erh1Δ derivative ZBM1020 transformed with pREP1 (ZBM1024), ZBM1020 transformed with pREP1/SpErh1p (ZBM1025), ZBM1020 transformed pREP1/SjErh1p (ZBM1026) and ZBM1020 transformed with pREP1/HsERH (ZBM1027). (B) Intracellular localization of SpErh1p in human HeLa cells by confocal microscopy. Upper series of images, cells transfected with plasmid coding for EGFP-tagged human ERH (pEGFP-N1/ER) alone or cotransfected with plasmid coding for mCherry-tagged human Ciz1 (pmCherry-N1/Ciz1); lower series of images, cells transfected with plasmid coding for EGFP-tagged SpErh1p (pEGFP-N1/SpErh1p) alone or cotransfected with pmCherry-N1/Ciz1. Direct fluorescence of EGFP or mCherry was observed in live cells. (C) Yeast two-hybrid analysis with SpErh1p used as bait. The host S. cerevisiae L40 cells coexpressing human ERH and Ciz1, human ERH and PDIP46/SKAR (both pairs as positive controls), SpErh1p and Ciz1, and SpErh1p and PDIP46/SKAR were lysed and the activity of the lacZ reporter gene (conversion of X-gal to a blue precipitate represented here as a strong gray color of lysed cells) was determined.
Table 1.
Fission yeast strains used in this study.