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J. Microbiol. Biotechnol. (2017), 27(3), 633–643 https://doi.org/10.4014/jmb.1610.10045 Review Research Article jmb Pro-Apoptotic Role of the Human YPEL5 Gene Identified by Functional Complementation of a Yeast moh1Δ Mutation Ji Young Lee1, Do Youn Jun1, Ju Eun Park1, Gi Hyun Kwon1, Jong-Sik Kim2, and Young Ho Kim1* 1 Laboratory of Immunobiology, School of Life Science and Biotechnology, College of Natural Sciences, Kyungpook National University, Daegu 41566, Republic of Korea 2 Department of Biological Sciences, Andong National University, Andong 36729, Republic of Korea Received: October 18, 2016 Revised: February 4, 2017 Accepted: February 6, 2017 First published online February 7, 2017 *Corresponding author Phone: +82-53-950-5378; Fax: +82-53-955-5522; E-mail: [email protected] pISSN 1017-7825, eISSN 1738-8872 Copyright © 2017 by The Korean Society for Microbiology and Biotechnology To examine the pro-apoptotic role of the human ortholog (YPEL5) of the Drosophila Yippee protein, the cell viability of Saccharomyces cerevisiae mutant strain with deleted MOH1, the yeast ortholog, was compared with that of the wild-type (WT)-MOH1 strain after exposure to different apoptogenic stimulants, including UV irradiation, methyl methanesulfonate (MMS), camptothecin (CPT), heat shock, and hyperosmotic shock. The moh1Δ mutant exhibited enhanced cell viability compared with the WT-MOH1 strain when treated with lethal UV irradiation, 1.8 mM MMS, 100 μM CPT, heat shock at 50°C, or 1.2 M KCl. At the same time, the level of Moh1 protein was commonly up-regulated in the WT-MOH1 strain as was that of Ynk1 protein, which is known as a marker for DNA damage. Although the enhanced UV resistance of the moh1Δ mutant largely disappeared following transformation with the yeast MOH1 gene or one of the human YPEL1-YPEL5 genes, the transformant bearing pYES2YPEL5 was more sensitive to lethal UV irradiation and its UV sensitivity was similar to that of the WT-MOH1 strain. Under these conditions, the UV irradiation-induced apoptotic events, such as FITC-Annexin V stainability, mitochondrial membrane potential (Δψm) loss, and metacaspase activation, occurred to a much lesser extent in the moh1Δ mutant compared with the WT-MOH1 strain and the mutant strain bearing pYES2-MOH1 or pYES2-YPEL5. These results demonstrate the functional conservation between yeast Moh1 and human YPEL5, and their involvement in mitochondria-dependent apoptosis induced by DNA damage. Keywords: Apoptosis, DNA damage, Drosophila Yippee, human YPEL5, metacaspase, S. cerevisiae MOH1 Introduction The Drosophila Yippee protein was initially identified, using the yeast two-hybrid method, as an intracellular protein that interacts with a blood protein (hemoline) from the moth Hyalophora cecropia [1]. Since then, orthologs of the Drosophila Yippee protein have been found in a wide range of eukaryotes, from yeast to human. During comprehensive human chromosome sequence analysis, a novel gene family consisting of five members (YPEL1 through YPEL5) possessing a high homology with the Drosophila Yippee gene was identified [2]. Based on comparison of the overall amino acid sequences of human YPEL family members and Drosophila Yippee, human YPEL1-YPEL4 shows 43.4-45.5% identity with Drosophila Yippee, and human YPEL5 shows 70.8% identity, indicating YPEL5 among the human YPEL family members is the most closely aligned with the Drosophila Yippee protein. Semiquantitative polymerase chain reaction revealed that transcripts for YPEL3 and YPEL5 were ubiquitously expressed in human tissues; however, those for YPEL1, YPEL2, and YPEL4 exhibited more restrictive expression patterns [2]. Immunofluorescence staining of African green monkey kidney fibroblast COS-7 cells showed that Ypel5 is localized in the nucleus and centrosome at interphase, whereas it relocates to the spindle pole, mitotic spindle, March 2017 ⎪ Vol. 27 ⎪ No. 3 634 Lee et al. and midbody during the M phase [2, 3]. Interestingly, human YPEL3, which was described as a small unstable apoptotic protein in murine myeloid cells [4], has been reported to be involved in growth suppression, causing cellular senescence in human cell lines, and its expression is regulated by the tumor suppressor protein p53, with lower expression in several human tumors [5-7]. These previous observations raised the possibility that human YPEL family proteins play an important role in the regulation of cell division and/or apoptosis; however, this prediction remains to be elucidated. Furthermore, it is uncertain whether the intracellular function of YPEL family proteins, which was predicted in the human orthologs, can be extended to other eukaryotic species, including yeasts. The budding yeast Saccharomyces cerevisiae ortholog MOH1 gene was identified as a gene (YBL049W) possessing sequence similarity to the Drosophila Yippee gene when sequencing and functional analysis were performed for a 32,560 bp segment of the left arm of S. cerevisiae chromosome II [1, 8]. Although analysis of the phenotype of S. cerevisiae deletion mutants can be an efficient approach for studying gene function [9-11], there have been no reports on phenotypic analysis of the mutant strain to determine MOH1 gene function. Furthermore, S. cerevisiae is a good eukaryotic model organism for the study of many fundamental biological processes, such as apoptotic cell death, cell cycle regulation, and DNA repair, which occur commonly in eukaryotic cells [12, 13]. In these contexts, we have decided to compare the phenotype of the moh1Δ mutant strain with that of the wild-type (WT)-MOH1 strain, and to determine whether the mutant phenotype can be recovered to the WT levels by complementation using plasmids expressing the human YPEL family genes. In a previous study, we found the human YPEL5 gene to be an interesting gene, in that its mRNA expression was easily detectable in unstimulated human peripheral T cells and then rapidly declined to undetectable level by 5 h following immobilized anti-CD3 activation [14]. Additionally, we observed that ectopic overexpression of the YPEL5 gene in human cervical carcinoma HeLa cells caused a significant reduction in cell proliferation to the level of 47% of the control, suggesting that YPEL5 might exert a suppressive effect on cell proliferation. However, the possible role of YPEL5 in induction of apoptosis, which led to the antiproliferative activity, and underlying cellular and molecular mechanism remain to be elucidated. As an attempt to obtain direct evidence for a pro-apoptotic role of human YPEL5, in the present study, we have employed a budding yeast S. cerevisiae mutant strain, J. Microbiol. Biotechnol. constructed by deletion of the MOH1 gene, and the WTMOH1 strain to compare their cytotoxic sensitivity to different apoptogenic stimulants such as UV irradiation, DNA-damaging drug, heat shock, and hyperosmotic shock. Additionally, we have examined whether transformation of the moh1Δ mutant with recombinant plasmids bearing the MOH1 gene or one of the YPEL family genes can abrogate the elevated resistance of the mutant strain against lethal UV irradiation, due to their pro-apoptotic contribution to the mitochondrial apoptotic pathway. Materials and Methods Reagents, Kits, Antibodies, Cells, and Media The camptothecin (CPT) and the DNA alkylating agent methyl methanesulfonate (MMS) were purchased from Sigma-Aldrich (USA). An ECL western blot kit was purchased from GE Healthcare Life Sciences (UK). The FITC-Annexin V apoptosis detection kit and JC-1 (5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolocarbocyanine iodide) were purchased from Invitrogen (USA). Anti-Ynk1 (antiNM23-H1) was purchased from Santa Cruz Biotechnology (USA), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody was purchased from Thermo Scientific (USA). Rat antisera raised against either recombinant human YPEL5 protein or recombinant S. cerevisiae Moh1 protein, both of which were expressed as GST-fusion forms using an Escherichia coli system, were prepared essentially as described previously [15]. These rat polyclonal anti-YPEL5 and anti-Moh1 antibodies were able to react with human YPEL5 and yeast Moh1, respectively. Wild-type (WT)-MOH1 S. cerevisiae BY4741 (MATa, his3∆1, leu2∆0, met15∆0, ura3∆0) and the MOH1-deletion mutant derived from S. cerevisiae BY4741 were obtained from ATCC (USA). The WT-MOH1 strain and the moh1Δ mutant strain were cultured in YPD medium containing 2% dextrose, 2% peptone, 1% yeast extract, and 200 μg/ml G418. The yeast strains overexpressing Moh1 or YPEL1-YPEL5 were constructed by transformation of the yeast moh1Δ mutant with recombinant plasmid pYES2-MOH1 or pYES2-YPEL1-pYES2-YPEL5. Both the WT-MOH1 strain and moh1Δ mutant were transformed with empty vector plasmid pYES2 and used as controls. Since the expression vector pYES2 contains the URA3 gene, individual transformants were selected on synthetic complete galactose (SC-gal) solid medium (2% galactose, 0.67% yeast nitrogen base w/o amino acid, 0.01% leucine, 0.005% methionine, 0.005% histidine, 200 μg/ml G418, and 1.8% agar). Treatment Conditions for UV Irradiation, DNA-Damaging Drug, Heat Shock, and Hyperosmotic Shock, and Cell Viability Assays To compare the sensitivity of the WT-MOH1 strain and the MOH1-deletion mutant with various cytotoxic factors, the cells grown in YPD medium at log phase were suspended in fresh YPD Apoptotic Activity of Yeast Moh1 and Human YPEL5 medium (A600 = 0.4). Five microliters of 3-fold serial dilutions of each cell suspension was plated on either YPD agar medium to expose to UV irradiation (180 J/m2) using a UV crosslinker (CL1000; Ultra-Violet Products Ltd., USA), or YPD agar medium containing 1.8 mM MMS, 100 μg/ml CPT, or 1.2 M KCl. To examine their sensitivity to heat shock, the cell suspension in fresh YPD medium (A600 = 0.4) was treated at 50°C for 20 min prior to serial 3-fold dilution and plating on YPD agar medium. To estimate the sensitivity of the WT-MOH1 strain- or the MOH1-deletion mutant-derived transformants to lethal UV irradiation, the individual cells grown in SC-gal liquid medium at log phase were resuspended in fresh SC-gal medium (A600 = 0.4). Five microliters of 3-fold serial dilutions of individual cell suspensions was plated on SC-gal solid medium. The uncovered plates were irradiated with UV light at a diverse range of doses. To examine quantitatively the effect of UV irradiation on cell viability, the individual cells were appropriately diluted and spread on SC-gal solid medium. After the uncovered SC-gal plates were exposed to UV irradiation, the plates were incubated at 30°C for 3 days and the colony numbers were counted to determine cell viability. An equivalent cell suspension in a fresh SC-gal medium (5 ml, A600 = 1.2) was spread on the surface of a 100-mm-diameter Petri dish and then irradiated with UV light at a diverse range of doses. Sequentially, the cells were incubated at 30°C with shaking on a rotary shaker at 120 rpm for indicated time periods for flow cytometric and western blot analyses, and cell viability assays. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) of Yeast MOH1 Gene and Human YPEL Family Genes, and Construction of Recombinant pYES2 Plasmids PCR amplification of human YPEL1-YPEL5 cDNA from a human T-cell cDNA library derived from Jurkat cell clone E6-1 (Stratagene, USA) was performed using the AccuPower PCR PreMix (Bioneer, Korea) and specific primers. The forward primers with BamHI sites and reverse primers with EcoRI sites used for PCR were YPEL1-forward, 5’-CGGATCCATGGTGAAAATGACAAAG-3’; YPEL1-reverse, 5’-TGAATTCTTACTCCCAGCCATTGTC-3’; YPEL2-forward, 5’-CGGATCCATGGTGAAGATGACAAGA-3’; YPEL2-reverse, 5’-AGAATTCTCAGTCCCAGCCATTGTC-3’; YPEL3-forward, 5’-CGGATCCATGGTGCGGATTTCAAA-3’; YPEL3-reverse, 5’-AGAATTCTCAGTCCCAGCCGTTGT-3’; YPEL4-forward, 5’-ATAGGATCCATGCCCAGCTGTGAC-3’; YPEL4-reverse, 5’-CGAATTCTCAGTCCCAGCCGTTGT-3’; YPEL5-forward, 5’-TCTAGGATCCATGGGCAGAATTTTC-3’; YPEL5-reverse, 5’-ACGGAATTCTCAAGAGTTATCAGATG-3’. For cloning of yeast MOH1 cDNA, total RNA was purified from the WT-MOH1 S. cerevisiae BY4741 strain as described previously [16]. The first-strand cDNAs were synthesized from 5 μg of total RNA using SuperScript II Reverse Transcriptase (Invitrogen) according to the manufacturer’s instructions. The MOH1-forward primer with BamHI site (5’-AGGATCCAATATGGGATTGCGTT-3’) and MOH1-reverse primer with EcoRI site (5’-GCGGAATTCTCA 635 AGTACATTTAC-3’) were used for the PCR. The amplified cDNA fragments of the individual transcripts were cloned into the BamH1-EcoRI site of pYES2 (Invitrogen), resulting in pYES2MOH1 and pYES2-YPEL1-pYES2-YPEL5, in which the cDNA was placed under a strong GAL1 promoter in sense orientation. Transformation of these recombinant plasmids into the moh1Δ mutant was performed as described previously [17]. To confirm the mRNA expression of individual cDNA inserts in the transformants, RT-PCR was performed using both forward and reverse primers. Additionally, for RT-PCR data normalization, the yeast housekeeping actin gene ACT1 (forward primer, 5’-AGGTTGCTGCTTTGGTTATTGATAACGG-3’; reverse primer, 5’-AGCCAAGATAGAACCACCAATCCAGACG-3’) was used. The PCR products were electrophoresed on a 1.2% agarose gel containing ethidium bromide and visualized under UV light. Sequence Analysis and Phylogenetic Tree Construction The amino acid sequences of the Drosophila Yippee, S. cerevisiae Moh1, S. pombe Ypel, and related Xenopus, mouse, and human YPEL family proteins were obtained by BLAST searches of the NCBI database (http://www.ncbi.nlm.nih.gov). The amino acid sequences of the Drosophila Yippee, S. cerevisiae Moh1, S. pombe Ypel, Xenopus Ypel1.L-Ypel5.L, mouse Ypel1-Ypel5, and human YPEL1-YPEL5 were aligned using Clustal X [18], and multiple alignments were manually edited in GeneDoc (Free Software Foundation, Inc., USA) to shade black for 100% conservation, gray for 80% conservation, and light gray for 60% conservation. The unrooted phylogenetic tree of the yeast Moh1 and individual human YPEL family members was constructed based on the amino acid sequence similarities using NJplot [19]. Yeast Cell Lysate and Western Blot Analysis Yeast cells (5 × 107) suspended in 1× SDS sample buffer were lysed by boiling for 10 min and then chilled on ice for 10 min. After centrifugation at 19,000 ×g for 20 min, the supernatant was collected as cell lysate. The cell lysates were subjected to electrophoresis on 12% SDS-polyacrylamide gels with Tris-glycine buffer, and then electrotransferred to nylon membranes. Western blot analysis was performed using the ECL western blotting detection system, as described previously [20]. Flow Cytometric Analysis For analysis of apoptosis, yeast cells were incubated in sorbitol buffer (0.6 M D-sorbitol, 10 mM β-mercaptoethanol, 20 mM TrisHCl, pH 7.4) containing Zymolyase 20T (20 unit/ml) at 30°C for 15 min to prepare protoplasts. Sequentially, the protoplasts (2 × 106) were washed with 1× binding buffer containing 0.6 M Dsorbitol, and treated with FITC-Annexin V and propidium iodide (PI) for 15 min prior to flow cytometric analysis (FACSCalibur; BD, USA). To measure intracellular metacaspase activation, yeast cells were stained with the CaspGLOW fluorescein active caspase staining kit (Biovision Inc., USA) prior to flow cytometric analysis according to the manufacturer’s instructions. Additionally, the cells March 2017 ⎪ Vol. 27 ⎪ No. 3 636 Lee et al. stained with FITC-VAD-FMK were examined and photographed using an LSM 700 confocal laser scanning microscope (Carl Zeiss MicroImaging GmbH, Germany). The mitochondria membrane potential (Δψm) loss of yeast cells was analyzed by flow cytometry using JC-1 dye that has the property of aggregating upon mitochondrial membrane polarization, forming an orangered fluorescence compound [20, 21]. Briefly, yeast cells were incubated with JC-1 dye (2 ng/ml in 1× PBS) at 30°C for 20 min, and the percentage of red and green fluorescence was measured using flow cytometry. Statistical Analyses Unless otherwise indicated, each result in this paper is representative of at least three separate experiments. Statistical analysis was performed using Students t-test to evaluate the significance of differences between two groups, and one-way ANOVA between three and more than three groups. In all graphs, * indicates p < 0.05 between the untreated and treated cells. All data are expressed as the mean ± standard deviation (SD; for each group n ≥ 3). One-way ANOVA followed by Dunnett’s multiple comparison test was also used for statistical analysis using IBM SPSS Statistics ver. 19. Results and Discussion Alignment of the Amino Acid Sequences of S. cerevisiae Moh1 and Human YPEL Family Proteins When the amino acid sequence of S. cerevisiae Moh1 was compared with those of human YPEL1-YPEL5 using the Clustal X program [18], the similarity of S. cerevisiae Moh1 (GenBank P38191) showed 38.7%, 37.8%, 37.8%, 40.2%, and 31.4% identity with YPEL1 (GenBank O60688), YPEL2 (GenBank Q96QA6), YPEL3 (GenBank P61236), YPEL4 (GenBank Q96NS1), and YPEL5 (GenBank P62699), respectively (Fig. 1A). At the same time, human YPEL3 shares 88.2%, 89.1%, and 79.0% identity with YPEL1, YPEL2, and YPEL4, respectively; however, YPEL5 shares 37.2%-42.0% identity with other human YPEL family members. The unrooted phylogenetic relationships of Drosophila Yippee protein, S. cerevisiae Moh1 protein, and human YPEL family proteins showed that Drosphila Yippee and S. cerevisiae Moh1 were grouped more closely with YPEL5 than with the other four YPEL family members (Fig. 1B). Consequently, these results suggest that the human ortholog of yeast Moh1 may be YPEL5, and cellular functions of YPEL1-YPLE4 could be different from that of YPEL5; however, their functional overlapping or distinction from each other remains uncertain. To examine whether the cellular function of S. cerevisiae Moh1 is associated with induction of apoptosis, the J. Microbiol. Biotechnol. cytotoxic sensitivity of the moh1Δ mutant was compared with that of the WT-MOH1 strain under various lethal apoptogenic conditions, such as UV irradiation, DNAdamaging drug, heat shock, and hyperosmotic treatments. When the cell viability levels of the moh1Δ mutant and the WT-MOH1 strains following UV irradiation at lethal doses were compared using the 3-fold serial dilution plate assay, the viability of the moh1Δ mutant was much higher than that of the WT-MOH1 strain, indicating that moh1Δ confers UV resistance (Fig. 2A). The enhanced resistance of the moh1Δ mutant compared with the WT-MOH1 strain was also observed following various apoptogenic treatments with 1.8 mM MMS, 100 μM CPT, heat shock at 50°C, and 1.2 M KCl, in which DNA damage-induced apoptosis can be mainly involved [22-25]. At the same time, western blot analysis revealed that the Moh1 protein level in the WTMOH1 strain was commonly enhanced at 3-6 h after exposure to these lethal treatments (Fig. 2B). The Ynk1 protein level was also elevated in common at 3-6 h. The yeast Ynk1 is known as a DNA damage marker, because Ynk1 is the yeast ortholog of human NM23-H1, possessing significant 3’-5’ exonuclease activity and nucleoside diphosphatase kinase activity, which were previously required for repair of DNA damage [26, 27]. Consequently, these previous and current results indicate that the Moh1 expression was up-regulated in the yeast S. cerevisiae under various lethal apoptogenic conditions to determine cytotoxic sensitivity, possibly through being involved in apoptotic cell death induced by DNA damage. Differential Sensitivities to Lethal UV Irradiation of S. cerevisiae WT-MOH1 Strain, moh1Δ Mutant, and Mutant-Derived Transformants Expressing MOH1 Gene or Individual YPEL Family Genes We decided to examine whether the mutant phenotype can be restored to the WT phenotype by complementation using recombinant pYES2 plasmids expressing the MOH1 gene or individual YPEL family genes. Thus, the response of the moh1Δ mutant to a lethal dose of UV irradiation was compared with those of the WT-MOH1 strain and the moh1Δ mutant-derived transformants bearing pYES2-MOH1 or pYES2-YPEL1-pYES2-YPEL5. As evidenced by RT-PCR analysis, the moh1Δ mutant failed to express the MOH1 mRNA transcript, whereas the individual transformants bearing pYES2-MOH1 or one of pYES2-YPEL1-pYES2YPEL5 appeared to overexpress each mRNA transcript compared with the WT-MOH1 strain (Fig. 3A). Under these conditions, the 3-fold serial dilution plate assay with UV irradiation at lethal doses of 130, 150, and 180 J/m2 showed Apoptotic Activity of Yeast Moh1 and Human YPEL5 637 Fig. 1. Comparison of amino acid sequences (A) and unrooted phylogenetic tree (B) of Drosophila Yippee (GenBank Q9XZF0), S. cerevisiae Moh1 (GenBank P38191), S. pombe Ypel (GenBank CAB62089), Xenopus Ypel1.L (GenBank NP_001086812), Xenopus Ypel2.L (GenBank NP_001089503), Xenopus Ypel5.L (GenBank NP_001087620), and mouse and human YPEL family members. Amino acids are displayed in single-letter abbreviation after alignment for maximal identity using Clustal X [18], and their phylogenetic relationships were constructed based on the amino acid sequence similarities using NJplot [19]. Hyphens represent introduced gaps for optimum alignment. Amino acid residue numbering is shown at the end of each line. By using the GeneDoc program (Free Software Foundation, Inc., USA), conserved residues are shaded black for 100% conservation, gray for 80% conservation, and light gray for 60% conservation. March 2017 ⎪ Vol. 27 ⎪ No. 3 638 Lee et al. Fig. 2. Comparison of the cell viability (A) and up-regulation of the protein levels of Moh1 and Ynk1 (B) of the WT-MOH1 strain and moh1Δ mutant strains treated with UV irradiation, methyl methanesulfonate (MMS), camptothecin (CPT), heat shock, and 1.2 M KCl. For analysis of the lethal effect of UV irradiation (180 J/m2), CPT (100 µg/ml), and heat shock (50°C for 20 min) on cell viability, the exponentially growing yeast strain was resuspended in fresh YPD medium and the 3-fold-serial dilution assay was performed as described in Materials and Methods. Equivalent cultures were prepared, harvested at the indicated time points, and subjected to western blot analysis. The yeast Ynk1 was used as the DNA damage marker. that the UV resistance of the moh1Δ mutant was significantly higher than that of the WT-MOH1 strain. However, the elevated UV resistance was abrogated when the mutant strain was transformed with pYES2-MOH1 (Fig. 3B). Interestingly, the transformation of the mutant strain with pYES2-YPEL1-pYES2-YPEL5 also led to abrogation of the elevated UV resistance with no significant difference in their abilities to restore normal UV sensitivity to the mutant strain. For quantitative analysis of the elevated UV resistance of the moh1Δ mutant strain, the individual cells were appropriately diluted, spread on SC-gal solid medium plates, and then exposed to lethal UV irradiation. After incubation at 30°C for 3 days, the colony numbers were J. Microbiol. Biotechnol. counted to calculate the cell viability. Although the cell viability of the moh1Δ mutant strain appeared to be ~130fold higher than that of the WT-MOH1 strain, this mutant phenotype disappeared following transformation with either the yeast MOH1 gene or one of the human YPEL family genes (Fig. 3C). Additionally, the transformant overexpressing the YPEL5 gene was more sensitive to lethality of UV irradiation than the other transformants overexpressing one of the YPEL1-YPEL4 genes, and its level of UV sensitivity was similar to that of the WT-MOH1 strain. These results demonstrate that the functional role of the S. cerevisiae MOH1 gene, which is crucial for determining UV sensitivity, can be complemented by human YPEL Apoptotic Activity of Yeast Moh1 and Human YPEL5 639 Fig. 3. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of the mRNA transcripts for the yeast Moh1 and human YPEL family proteins in the WT-MOH1 strain, moh1Δ mutant strain, and mutant-derived transformants expressing the MOH1 gene or each human YPEL gene (A); UV resistance test of the yeast strains using the 3-fold serial dilution plate assay (B); and viable cell counts with the spreadplate method (C). Individual yeast strains were grown in SC-gal liquid medium, harvested at log phase, and subjected to isolation of total RNA. RT-PCR was performed as described in Materials and Methods. For RT-PCR data normalization, the yeast housekeeping actin gene ACT1 was used. To investigate the cytotoxic effect of UV irradiation, both the 3-fold serial dilution assay and the spread-plate method were used as described in Materials and Methods. After the uncovered plates were exposed to UV irradiation at the indicated doses, the plates were incubated at 30°C for 3 days before measuring cell viability. Each value is expressed as the mean ± SD (n = 3). *p < 0.05 compared with the control. March 2017 ⎪ Vol. 27 ⎪ No. 3 640 Lee et al. family genes. Additionally, these results suggest that although the functional roles of human YPEL family genes overexpressed in S. cerevisiae are overlapping with the yeast MOH1 gene, the YPEL5 gene is more likely to be the human ortholog of the yeast MOH1 gene. Pro-Apoptotic Role of Yeast Moh1 and Human YPEL5 Proteins in UV-Induced Apoptotic Pathway The basic apoptosis program is known to be present in the yeast S. cerevisiae cells, in that the heterologous expression of human Bax in yeast cells provokes several apoptotic events, including phosphatidylserine externalization at the cell surface, cytochrome c release, and DNA fragmentation, and that the concomitant expression of human Bcl-xL prevents these effects and cell death [28-31]. Because our data indicated a contribution of the yeast MOH1 gene as a determinant of UV sensitivity, and the functional complementation of yeast MOH1 gene with its orthologous human YPEL5 gene, we examined whether yeast Moh1 and human YPEL5 can play a pro-apoptotic role in the UVinduced apoptotic pathway. In accordance with the RT-PCR data, western blot analyses also showed that the moh1Δ mutant strain failed to express the Moh1 protein; however, the WT-MOH1 strain and the transformants bearing pYES2-MOH1 and pYES2YPEL5 successfully expressed yeast Moh1 and human YPEL5, respectively (Fig. 4A). It is noteworthy that the Moh1 level in the transformant appeared to be 24.5-fold higher than that in the WT-MOH1 strain, demonstrating overexpression of Moh1 in the transformant bearing pYES2-MOH1. Under these conditions, four strains (the WT-MOH1 strain, moh1Δ mutant strain, and transformants expressing yeast MOH1 gene or human YPEL5 gene), which were incubated for 6 h following UV irradiation (180 J/m2), were converted into protoplasts and analyzed by FITC-Annexin V and PI staining to detect apoptotic Fig. 4. Western blot analyses of the S. cereviasie Moh1 protein and human YPEL5 proteins in the WT-MOH1 strain, moh1Δ mutant strain, and each transformant bearing pYES2-MOH1 or pYES2-YPEL5 (A); flow cytometric analyses of apoptotic cells using Annexin V-FITC and propidium iodide (PI) staining (B); and flow cytometric analyses of Δψm loss using JC-1 dye (C) in individual yeast strains following lethal UV exposure. After exponentially growing individual yeast strains were resuspended in a fresh SC-gal medium (A600 = 0.6), exposed to UV irradiation (180 J/m2), and then incubated at 30°C with shaking for 6 h, the cells harvested were subjected to western blot and flow cytometric analyses, as described in Materials and Methods. J. Microbiol. Biotechnol. Apoptotic Activity of Yeast Moh1 and Human YPEL5 cells. After treatment of the WT-MOH1 strain with UV irradiation, the rate of apoptotic cells was enhanced to 34.6% (14.5% early apoptotic cells stained only with FITCAnnexin V; 20.1% late apoptotic cells stained with both FITC-Annexin V and PI), whereas the moh1Δ mutant strain exhibited only 11.1% apoptotic cells (9.0% early apoptotic cells; 2.1% late apoptotic cells), demonstrating that the moh1Δ mutation confers resistance to the apoptogenic effect of UV irradiation (Fig. 4B). Under these conditions, the rates of UV-induced apoptotic cells for the transformants expressing Moh1 and YPEL5 were 44.3% (21.6% early apoptotic cells; 22.7% late apoptotic cells) and 31.9% (16.8% early apoptotic cells; 15.1% late apoptotic cells), respectively. These results indicate that yeast Moh1 plays a proapoptotic role in the UV-induced apoptotic pathway, so its deletion mutation could confer UV resistance, and that the pro-apoptotic role of Moh1 protein in yeast cells is functionally complemented by human YPEL5 protein. The Δψm loss, mitochondrial cytochrome c release into the cytosol, and subsequent caspase cascade activation are hallmark events of apoptosis [32]. To understand which modulator of the apoptotic pathway can be influenced by the pro-apoptotic activity of yeast Moh1 and human 641 YPEL5, the change in Δψm of the four strains following exposure to lethal UV irradiation was measured. The UV irradiation caused a significant Δψm loss in the WT-MOH1 strain and transformants bearing pYES2-MOH1 or pYES2YPEL5, with the exception of the MOH1-deletion mutant strain (Fig. 4C). Since Δψm disruption is known to be one of the initial intracellular changes leading to apoptotic cell death [33], these results suggest that the pro-apoptotic role of yeast Moh1 and human YPEL5 was involved in UVinduced Δψm disruption in yeast cells. In addition, flow cytometric analysis of intracellular metacaspase activation using FITC-VAD-FMK, which detects the activation of metacaspase in yeast cells [34], revealed that the rates of cells with UV-induced metacaspase activation for the WT-MOH1 strain, moh1Δ mutant, transformant bearing pYES2-MOH1, and transformant bearing pYES2YPEL5 were 67.3%, 22.1%, 85.8%, and 78.9%, respectively (Figs. 5A and 5B). Fluorescence microscopic analysis also showed a markedly lower rate of metacaspase activation in the moh1Δ mutant compared with WT-MOH1 strain and transformants bearing pYES2-MOH1 or pYES2-YPEL5. These results demonstrate that the pro-apoptotic activity of yeast Moh1 protein, which can be complemented by Fig. 5. Flow cytometric and fluorescence microscopic analyses of metacaspase activation in the WT-MOH1 strain, moh1Δ mutant strain, and each transformant bearing pYES2-MOH1 or pYES2-YPEL5 following none treatment (A) and lethal UV exposure (B). After exponentially growing individual yeast strains were resuspended in a fresh SC-gal medium (A600 = 0.6), exposed to UV irradiation (180 J/m2), and then incubated at 30°C with shaking for 6 h, the cells harvested were subjected to flow cytometric and fluorescence microscopic analyses, as described in Materials and Methods. The scale bar represents a length of 10 µm in each of the images. March 2017 ⎪ Vol. 27 ⎪ No. 3 642 Lee et al. human YPEL5, is a prerequisite for inducing mitochondrial damage and subsequent activation of metacaspase during UV-induced apoptotic cell death in S. cerevisiae. 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