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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,
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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
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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.
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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
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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.
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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.
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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.
In conclusion, these results demonstrate that S. cerevisiae
Moh1 protein, which is the ortholog of Drosophila Yippee
protein, is a pro-apoptotic modulator crucial for Δψm loss
and resultant activation of metacaspase during UVinduced apoptosis, and its deletion mutation results in UV
resistance. The pro-apoptotic role of the Moh1 protein in
yeast cells is functionally complemented by human YPEL
family proteins YPEL1-YPEL5, with the highest efficiency
by YPEL5. Additionally, the moh1Δ mutant shows enhanced
resistance compared with the WT-MOH1 strain to different
apoptogenic treatments, all of which are known to cause
DNA damage-induced apoptosis, suggesting the conservation
of a pro-apoptotic role of yeast Moh1 and human YPEL5 in
DNA damage-induced apoptosis.
Acknowledgments
This study was supported by the Basic Science Research
Program through the National Research Foundation of
Korea (NRF) funded by the Ministry of Education (NRF2013R1A1A2065403).
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