Small Toxic Protein Encoded on Chromosome VII of Saccharomyces cerevisiae
Small Toxic Protein Encoded on Chromosome VII of Saccharomyces cerevisiae
Koji Makanae 0 1
Reiko Kintaka 0 1
Koji Ishikawa 0 1
Hisao Moriya 0 1
Stephan Neil Witt, Louisiana State
University Health Sciences Center, UNITED STATES
0 1 Research Core for Interdisciplinary Sciences, Okayama University , Kita-ku, Okayama , Japan , 2 Graduate School of Science and Technology, Okayama University , Kita-ku, Okayama , Japan
1 Data Availability Statement: All RNAseq data files are available from the DDBJ Sequence Read Archive (accession number DRA002585)
In a previous study, we found an unknown element that caused growth inhibition after its copy number increased in the 30 region of DIE2 in Saccharomyces cerevisiae. In this study, we further identified this element and observed that overexpression of a small protein (sORF2) of 57 amino acids encoded in this region caused growth inhibition. The transcriptional response and multicopy suppression of the growth inhibition caused by sORF2 overexpression suggest that sORF2 overexpression inhibits the ergosterol biosynthetic pathway. sORF2 was not required in the normal growth of S. cerevisiae, and not conserved in related yeast species including S. paradoxus. Thus, sORF2 (designated as OTO1) is an orphan ORF that determines the specificity of this species.
We previously analyzed the copy number limits of most of the protein-coding genes in the
budding yeast Saccharomyces cerevisiae using the genetic tug-of-war (gTOW) method . In the
gTOW method, the copy number of a plasmid containing a target gene (with its native promoter
and terminator region) is increased on basis of the selection bias of the leu2d gene [2,3]. The
copy number of the empty plasmid exceeds 100 in the leucine-negative condition. If the target
gene has a copy number limit of <100, the plasmid copy number reflects the copy number limit.
When a target gene has the low copy number limit, we consider that overexpression of the
protein encoded by the target gene (i.e., the annotated open reading frame (ORF)) results in
growth inhibition. However, elements other than the target gene in the DNA fragment could
determine the low copy number. For example, increasing the copy number of a DNA element,
overexpression of an RNA element, or overexpression of an unannotated protein could result
in growth inhibition.
To test this possibility, we previously analyzed the low limit genes by introducing a frameshift
mutation to disrupt each annotated ORF and we isolated 10 DNA fragments where frameshift
mutations in the annotated ORFs still obtained low copy number limits . We also dissected
the fragments and isolated four DNA fragments with unknown elements that determined the
low copy number limits. Thus, we isolated a 600-base pair (bp) DNA fragment that contained
the 30 region of DIE2, which resulted in a low copy number limit (Frag5 in Fig. 1A) .
Competing Interests: The authors have declared
that no competing interests exist.
In this study, we further analyzed this region and showed that expression of a small ORF
encoding 58 codons caused growth inhibition.
To isolate the specific element responsible for the low copy number limit in the 30 region of
DIE2, we introduced a series of 10-bp deletions in every 100 bp of Frag5 and measured their
copy number limits. As shown in Fig. 1B, deletions of two sites in the downstream region of
DIE2 increased the copy number limit to >100. As shown in Fig. 1C, two small ORFs of >100
bp are encoded in Frag5 (denoted as sORF1 and sORF2). Both of these two 10-bp deletions
disrupted sORF2, which indicates that sORF2 might be responsible for the low copy number limit
To disrupt sORF2 alone, we introduced mutations to change the potential start codons
(ATG) of sORF2 into ATC. The results obtained are shown in Fig. 1D. Frag5 with a mutation
that changed the first ATG codon of sORF2 into ATC possessed a copy number limit of >100.
Frag5 with a mutation in the second ATG had a higher limit than the original Frag5, but the
limit was still low (28.6 3.5). This result strongly suggests that overexpression of the protein
encoded by sORF2 causes growth inhibition when its copy number is increased in the 30 region
of DIE2. Fig. 1E shows the amino acid sequence of sORF2.
High level expression of sORF2 driven by the GAL1 promoter inhibits
To confirm whether sORF2 overexpression alone caused growth inhibition, we tried to express
sORF2 from the GAL1 promoter (PGAL1). As shown in Fig. 2A, yeast cells that harbored the
PGAL1sORF2 plasmid did not grow on galactose plates. Next, we observed the growth
inhibition process using time-lapse microscopic imaging. As shown in Fig. 2B, at the time point
when the induction of PGAL1-GFP was observed, each cell that expressed sORF2 ceased its
proliferation and a large void structure was present. These results indicate that the high level
expression of sORF2 inhibited cellular growth.
sORF2 is not required for the normal growth of S. cerevisiae
To test whether sORF2 is required for the growth of S. cerevisiae, we disrupted sORF2 by
replacing it with a kanamycin resistance gene cassette (KanMX), as shown in Fig. 2C. The
sORF2::KanMX cells exhibited the same growth as the wild-type cells in normal growth
conditions (YPD, 30C; Fig. 2C).
Increasing the copy number of the sORF2-containing DNA fragment
induces the expression of ergosterol synthesis genes
We performed transcriptome analysis (RNAseq) to analyze the cellular response after the
overexpression of sORF2. We compared the mRNA expression profiles of cells that harbored the
vector plasmids and the plasmid containing the DIE2 30 fragment (Rear2, Fig. 1A).
Tables 1 and 2 show the genes with significantly different expression levels.
We analyzed the enriched genes based on gene ontology (GO) terms. The genes with higher
expression levels in the cells that harbored the pTOW-Rear2 plasmid were significantly
enriched in terms of genes involved in the ergosterol biosynthesis pathway (p = 2.2e4). Eight
genes (DAN1, DAN4, ERG1, ERG3, ERG11, ERG25, TIR3, and TIR4) with higher expression
Fig 1. Isolation of the element responsible for the low copy number limit in the DIE2 region.
A. Copy number limits of DNA fragments from the DIE2 region. The data were obtained from our previous study .
B. Copy number limits of DNA fragments (Frag5 in A) with serial 10-bp deletions every 100 bp. The asterisk indicates that only single experiment
C. Locations of the small ORFs (sORF1 and sORF2) in the 30 region of DIE2. The numbers indicate the 10-bp deletions analyzed in B. The deletions shown in
white did not affect the toxicity of the DNA fragment, whereas the deletion shown in black disrupted the toxicity.
D. Copy number limits of DNA fragments with ATG to ATC substitutions in sORF2.
E. Amino acid sequence of sORF2. The substituted methionines (ATG codons) in C are shown in red. A potential NLS sequence is underlined, and an amino
acid sequence predicted to construct a helical structure is shown in bold letters.
Fig 2. Genetic analyses of sORF2.
A. Overexpression of sORF2 from the GAL1 promoter (PGAL1). The construct used in this experiment is shown. Cells with pTOW-PGAL1-sORF2
(PGAL1sORF2) were streaked onto SC-glucose and SC-galactose plates. Two independent plasmid clones were analyzed. pTOW40836 (Vector) was used as
an empty vector control and pTOW-PGAL1-GFP (PGAL1-GFP) was used to monitor the PGAL1 induction.
B. Time-lapse imaging of cells after the induction of sORF2. The cells with pTOW-PGAL1-sORF2 (PGAL1-ORF2) and pTOW-PGAL1-GFP (PGAL1-GFP) were
cultured in SC-glucose mixed at a ratio of 10:1 and then cultivated in SC-galactose medium. PGAL1-GFP was used to monitor the induction of PGAL1. The
cellular images shown were obtained every 5min. A movie is available as S1 Movie.
C. Deletion of sORF2. The construct used to delete sORF2 from the chromosome is shown. The strain with sORF2 deleted was streaked onto a YPD agar
plate. The strain BY4741 was used as a wild-type control.
*Saccharomyces genome database: http://www.yeastgenome.org
levels were identified as genes that could be induced by treatment with ketoconazole .
Ketoconazole is known to inhibit the ergosterol biosynthetic pathway ; thus, sORF2
overexpression appeared to affect this pathway. The genes with lower expression levels were not
significantly enriched with respect to GO terms. They however contained many genes encoding
transporters and membrane proteins, such as ADY2, ENA1, FMP43, FMP45, HXT6, HXT7,
JEN1, PHO89, SMA1, and YNL194C, suggesting that sORF2 overexpression modulates the
expression of membrane proteins.
Expression analysis of sORF2
We analyzed the RNAseq data to determine whether sORF2 is transcribed. As shown in
Fig. 3A, transcript reads containing sORF2 were not detected in the mRNAs from BY4741 that
harbored an empty vector pTOWug2836, whereas a large number of transcript reads were
detected in the mRNAs that harbored pTOW-Rear2.
To test whether sORF2 was translated, we attached the tandem affinity purification (TAP)
tag to sORF2 and attempted to detect the TAP-tagged sORF2 by Western blotting. As shown in
Fig. 3B, sORF2-TAP expressed from its genomic region was detected, and the expression of
sORF2-TAP from the plasmid was highly increased. The expression of sORF2 from its
genomic region was detected in the log phage cell lysate, but not in the post-log phase lysate
(S1 Fig.). The expression was not increased under mating conditions (S1 Fig.).
We further estimated the expression level of sORF2-TAP in comparison to the expression
level of a reference protein Pop5-TAP, whose protein copy number was previously determined
(2230 copies/cell) . As the result, the expression level of sORF2-TAP from its genomic
region was estimated to be 45 copies/cell, which corresponds to the level of lowly expressed
proteins . The estimated expression level of sORF2-TAP from the plasmid was 1938 copies/cell.
*Saccharomyces genome database: http://www.yeastgenome.org
It should be noted that the copy number limit of the plasmid that contained the sORF2-TAP
DNA fragment was >100 (data not shown). This suggests that the small size of sORF2 itself is
required to inhibit growth.
Currently, we do not know the reason why we could not detect the mRNA of sORF2
expressed from its genomic region by our RNAseq analysis above. Although it is possible that
integrating TAP-tag sequence and a marker gene stimulated the expression of sORF2, the result
still suggests that there is an expression potential from the sORF2 locus. Supporting this idea,
there is a TA repeat in the upstream region of sORF2, which provides potential binding sites
for transcriptional factors such as the TATA-binding protein Spt15 (S2 Fig.). Notably, the TA
repeat is far shorter in the corresponding genomic region of S. paradoxus, which lacks sORF2
(Fig. 4A). These binding sites might function as promoters for sORF2.
Multicopy UBP7 and PRM1 suppress the growth inhibition caused by the
high copy number sORF2-containing DNA fragment
To further elucidate the molecular mechanism responsible for growth inhibition by sORF2, we
attempted to isolate multicopy suppressors of the growth inhibition caused by high copy
Fig 3. Expression analysis of sORF2.
A. RNAseq analysis of the sORF2 region of the strain BY4741 with the control vector (pTOWug2836) and pTOW-Rear2. Parts of the detected reads are
shown. The locations of DIE2, sORF2, and SMI1 are indicated.
B. Western blot analysis of sORF2 using TAPtag. Expression of sORF2-TAP from the genomic region or plasmids was detected using peroxidase
anti-peroxidase soluble complex. BY4714 is a negative control strain without any TAP-tagged protein expressed. Vector is another negative control, in which
BY4741 harbors an empty vector (pTOWug2836). Cells of BY4741, sORF2-TAP (genome), and POP5-TAP (genome) were cultivated in YPD medium;
cells of Vector and sORF2-TAP (plasmid) were cultivated in SCUra medium. Dilution indicates the fold-dilution of the cellular lysate applied to the gel.
Red-squared dilutions were used to calculate the expression levels of TAP-tagged proteins. The white arrowhead indicates the expected molecular weight
of Pop5-TAP protein (39.6kDa), and the black arrowhead indicates the one of sORF2-TAP (27.1 kDa). Structures of sORF2-TAP constructs are shown.
Fig 4. Structural analysis of sORF2.
A. Alignment of the sORF2 regions of S. cerevisiae and S. paradoxus. Identical nucleotides are shown in yellow. ATG and STOP codons of sORF2 are
shown in red. A TATA repeat and deletion in the S. paradoxus sequence are indicated in blue. The image is a snapshot from the fungal sequence
alignment of SGD (http://www.yeastgenome.org/cache/fungi/YGR229C.html). The nucleotide numbers indicate the positions relative to the stop codon of
B. Overexpression of sORF2 without the potential NLS (sORF2 KKRK). The construct used in this experiment is shown. Cells with pTOW-PGAL1-sORF2
(PGAL1-sORF2) or pTOW-PGAL1-sORF2KKRK (PGAL1-sORF2KKRK) were streaked onto SC-glucose and SC-galactose plates and incubated for indicated
days. pTOW40836 (Vector) was used as an empty vector control and pTOW-PGAL1-GFP (PGAL1-GFP) was used to monitor the PGAL1 induction.
A. Maximum growth rate of BY4741 cells that harbored both pTOW-Rear2 and the suppressor plasmids
(pRS423-UBP7 and pRS423-PRM1, and the empty vector, pRS423) in SCUraHis medium. The
averages and standard deviations from six independent experiments are shown.
B. Growth curves of the BY4741 cells that harbored both pTOW-Rear2 and the suppressor plasmids in SC
UraHis medium. One representative data is shown from each experiment.
number pTOW-Rear2. As shown in Fig. 5, we isolated two multicopy suppressors, UBP7 and
PRM1. UBP7 encodes a ubiquitin protease (UBPs) that controls protein degradation . PRM1
encodes a pheromone-regulated membrane protein, which is involved in membrane fusion
during mating . PRM1 is known to have a genetic interaction with ERG genes [9,10]. This
result also suggests the involvement of sORF2 in the ergosterol biosynthetic pathway.
Structural analysis of sORF2
In order to speculate the molecular function of sORF2, we performed some bioinformatics
analyses. We first performed the BLAST search toward the protein sequences stored at NCBI
database (http://blast.ncbi.nlm.nih.gov/), but we could not obtain any significantly
The corresponding ORF was not conserved in any closely-related yeast species (S.
paradoxus, S. bayanus, S. mikatae, S. castellii, and S. kudriavzevii). Fig. 4A shows the comparison of
the corresponding genomic locus from S. cerevisiae and S. paradoxus (most closely-related
species to S. cerevisae), as an example.
During the structural analysis, we noticed that sORF2 contained a consensus sequence of
nuclear localization signals (K-K/R-X-K/R)  at its C-terminal (underlined in Fig. 1E). To
test if this potential nuclear localization signal (NLS) is important for the toxicity of sORF2, we
overexpressed sORF2 without the sequence (sORF2KKRK). As shown in Fig. 4B, yeast cells
that harbored the PGAL1sORF2KKRK plasmid grew on galactose plates, but much slower than
the cells with the empty vector or PGAL1GFP plasmids. This result indicates that the potential
NLS is partly required (but not essential) for the toxicity of sORF2.
We next tried to predict the secondary and tertiary structure of sORF2 using a protein
homology/analogy recognition engine, Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2/). The
analysis predicted that there was a helical structure in the middle of the protein (shown in bold
letters in Fig. 1E) based on its similarity with two template proteins (d1k78a1 and d6paxa1)
with the confidence scores > 70 (the prediction results are summarized in S3 Fig.). Because the
template proteins were both structurally classified into DNA/RNA-binding 3-helical bundle
(Fold), homeodomain-like (superfamily), and paired domain (family), sORF2 might have
DNA/RNA binding activity.
sORF2 (OTO1/YGR228C-A) as an orphan ORF
In this study, we obtained evidence that overexpression of a small ORF of 58 codons (sORF2)
encoded within the 30 region of DIE2 causes growth inhibition. Our results also suggest that
sORF2 overexpression affects the ergosterol synthetic pathway. Based on the fact that sORF2
has a potential NLS and a helical structure involved in DNA/RNA binding, sORF2 might
function through its nuclear function such as transcriptional regulation.
sORF2 was not identified in previous studies that aimed to detect small ORFs based on their
expression and evolutionary conservation . In fact, sORF2 is not conserved in the
corresponding genomic region of the most closely-related yeast species S. paradoxus (Fig. 4A). We
thus think that sORF2 is an orphan ORF (ORFan) [16, 17], which distinguishes species by
functioning in species-specific cellular situations, and propose its name as OTO1 (ORFan toxic
when overexpressed) with its locus name YGR228C-A.
Our gTOW approach might be useful for isolating other ORFans. In fact, we had isolated
three more genomic loci potentially contain unannotated toxic elements when the copy
numbers were increased .
Materials and Methods
Strains and growth conditions
BY4741 (MATa his30 leu2 0 met15 0 ura3 0)  was used as the host yeast strain to test
the toxicity of DIE2 fragments and sORF2. The sORF2 deletion strain was created, as follows:
The genomic region of sORF2 (from ATG to stop) in BY4743 (MATa/ his3 1/his3 1 leu2
0/leu2 0 LYS2/lys2 0 met15 0/MET15 ura3 0/ura3 0)  was replaced by the
KanMX6 cassette using a DNA fragment, which was amplified by PCR with the primers
OHM0969 and OHM0970 using pKT127  as a template. The strains were sporulated, and
the tetrads were dissected. After genotypic analysis of the tetrads, haploid deletion strains were
isolated. The sORF2-TAP strain was created, as follows: A sORF2-TAP fragment was amplified
by PCR with the primers OHM1030 and OHM1032 using pTOW-sORF2-TAP. A hphMX4
fragment was amplified by PCR with primers the OHM1031 and OH1033 using pAG34 .
Both fragments were introduced into BY4741 to integrate sORF2-TAP-hphMX4 into the
genomic region of sORF2. BY4742 (MAT his31 leu20 lys20 ura30)  was used for a mating
partner of BY4741 with sORF2-TAP-hphMX4.
Yeast cells were grown in standard growth conditions . The PCR primers used to
amplify the DNA fragments employed in strain construction are listed in S1 Table.
Plasmids used in this study
The plasmids used in this study are listed in Table 3. The plasmids were constructed on the
basis of the homologous recombination activity of yeast cells . The PCR primers used to
amplify the DNA fragments, which were employed in plasmid construction are listed in
Measurement of the plasmid copy number limit
The copy number limits of plasmids were measured as described in our previous study .
Briefly, DNA from yeast cells grown in SCUra, SCUraLeu, or SCUraHis medium
were extracted, and the relative plasmid copy number compared with the genomic DNA in the
DNA solution was measured using real-time PCR. HIS3, LEU2, and LEU3 genes were detected
as indicators of the plasmid copy number for pRS423ks, pTOWug2836/40836, and genomic
DNA, respectively. More than two independent experiments were performed for each
experiment otherwise stated.
Yeast cells that harbored pTOWug2836, pTOW40836, and pTOW-Rear2 were cultivated in
SCUra medium until the mid-log phase, and RNA from each culture was then isolated using
the hot phenol method . A cDNA library was prepared using a SureSelect strand-specific
RNA library preparation kit (G9691A, Agilent), and sequencing was performed using an
Illumina Hiseq2500 with TruSeq SBS kit v3-HS. The software connected to GenomeSpace
(http://www.genomespace.org) was used for the sequence data analysis. The sequence data were
analyzed using TopHat (ver. 6) and Cufflink/cuffdiff (ver.4) on the GenePattern platform
(http://genepattern.broadinstitute.org), with sacCer3 for gene annotation (http://
genome.ucsc.edu/cgi-bin/hgTables). First, we isolated genes that differed significantly (FDR <
0.5) between pTOWug2836 and pTOW-Rear2 (Comp1), pTOW40836 and pTOW-Rear2
(Comp2), pTOWug2836 and pTOW40836 (Comp3), and the pTOW-Rear2 duplicates
(Comp4). Next, we prepared a gene list from genes isolated in Comp1 or Comp2, but not in
Comp3 or Comp4 (summarized in S4 Fig. and S2 Table). The Integrative Genomics Viewer
(IGV2.3, http://www.broadinstitute.org/igv/) was used to visualize the sequence reads shown in
Fig. 3A. The GO, publication, and pathway enrichments were analyzed using YeastMine
Cells were cultivated in SCUra medium until the mid-log phase and the cells were then
transferred to SC-galactoseUra medium, before being applied to a PDMS microfluidic
chamber (YC-1, Warner instruments). Cellular images were acquired every 5 min using a Leica
DM6000 B microscope. GFP fluorescence was determined using a GFP filter cube (excitation
filter 470/40 and emission filter 525/50).
Western blot analysis
Western blotting was performed as described previously . Briefly, proteins extracted from
the 0.25 OD600 cells (with indicated fold dilutions) cultivated in the indicated medium were
separated by SDS-PAGE and transferred onto a PVDF membrane. The TAP-tagged protein
was then detected using peroxidase anti-peroxidase soluble complex (P1901l, Sigma-Aldrich).
The chemiluminescent image was taken and the intensity of each band was measured using the
LAS-4000 image analyzer (GE Healthcare).
Multicopy suppressor screening
A multicopy plasmid library where most of the genes in S. cerevisiae were cloned into
pRS423ks (our laboratory stock) was introduced into yeast strains that harbored
pTOWRear2. Next, the colonies were grown on SCUraHis plates and then replica-plated onto SC
UraLeuHis plates. The plasmids were recovered from the colonies grown on SCUra
LeuHis and the DNA sequences of inserts in the plasmids were determined. The suppressor
activities of the isolated candidates were re-evaluated by measuring the growth of the cells that
harbored both pTOW-Rear2 and the suppressor plasmids in SCUraHis medium. Cellular
growth was measured by monitoring OD595 every 30 min using a microplate reader (Infinite
F200, TECAN). The maximum growth rate was calculated as described previously [2, 3].
S1 Fig. Expressions of sORF2-TAP from its genomic region in different conditions.
Expression of sORF2-TAP from the genomic region under indicated conditions were detected using
peroxidase anti-peroxidase soluble complex. Cellular lysates from the 0.0625 OD600 cells were
loaded. To create mating conditions, BY4741 with sORF2-TAP-hphMX4 cells were mixed with
BY4742 cells on a YPD agar plate and incubated for 2 hours in prior to prepare of the cellular
lysate. BY4741 with sORF2-TAP-hphMX4 cells were cultivated in YPD medium to prepare log
phase cells and post-log phase cells. The cellular density of the cultures were shown as OD600.
S4 Fig. Isolation of genes whose expressions were significantly changed upon increase in
DIE2-Rear2 fragment. We first isolated genes showing significant difference (FDR < 0.5)
between; pTOWug2836 and pTOWug2-Rear2 (Comp1), pTOW40836 and pTOWug2-Rear2
(Comp2), pTOWug2836 and pTOW40836 (Comp3), and between pTOWug2-Rear2
duplicates (Comp4). We then made a gene list, which contained true positives, from isolated genes
in Comp1 or Comp2, but neither in Comp3 nor Comp4 (see the Venn diagram).
Conceived and designed the experiments: HM. Performed the experiments: KM RK. Analyzed
the data: HM KI. Wrote the paper: HM.
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