Small Toxic Protein Encoded on Chromosome VII of <i>Saccharomyces cerevisiae</i>

<div><p>In a previous study, we found an unknown element that caused growth inhibition after its copy number increased in the 3′ region of <i>DIE2</i> in <i>Saccharomyces cerevisiae</i>. 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 <i>S</i>. <i>cerevisiae</i>, and not conserved in related yeast species including <i>S</i>. <i>paradoxus</i>. Thus, sORF2 (designated as <i>OTO1</i>) is an orphan ORF that determines the specificity of this species.</p></div

Isolation of the element responsible for the low copy number limit in the <i>DIE2</i> region.

<p></p><p></p><p></p><p>Copy number limits of DNA fragments from the <i>DIE2</i> region. The data were obtained from our previous study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120678#pone.0120678.ref001" target="_blank">1</a>].</p><p></p><p></p><p>Copy number limits of DNA fragments (Frag5 in A) with serial 10-bp deletions every 100 bp. The asterisk indicates that only single experiment was performed.</p><p></p><p></p><p>Locations of the small ORFs (<i>sORF1</i> and <i>sORF2</i>) in the 3′ region of <i>DIE2</i>. 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.</p><p></p><p></p><p>Copy number limits of DNA fragments with ATG to ATC substitutions in <i>sORF2</i>.</p><p></p><p></p><p>Amino acid sequence of <i>sORF2</i>. 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.</p><p></p><p></p><p></p

Structural analysis of sORF2.

<p></p><p></p><p></p><p>Alignment of the <i>sORF2</i> regions of <i>S</i>. <i>cerevisiae</i> and <i>S</i>. <i>paradoxus</i>. Identical nucleotides are shown in yellow. ATG and STOP codons of <i>sORF2</i> are shown in red. A TATA repeat and deletion in the <i>S</i>. <i>paradoxus</i> sequence are indicated in blue. The image is a snapshot from the fungal sequence alignment of SGD (<a href="http://www.yeastgenome.org/cache/fungi/YGR229C.html" target="_blank">http://www.yeastgenome.org/cache/fungi/YGR229C.html</a>). The nucleotide numbers indicate the positions relative to the stop codon of <i>SMI1</i>.</p><p></p><p></p><p>Overexpression of sORF2 without the potential NLS (sORF2 _ΔKKRK). The construct used in this experiment is shown. Cells with pTOW-P_GAL1-sORF2 (<i>P</i>_<i>GAL1</i><i>-sORF2</i>) or pTOW-P_GAL1-sORF2_ΔKKRK (<i>P</i>_<i>GAL1</i><i>-sORF2</i>_{<i>ΔKKRK</i>}) 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-P_GAL1-GFP (<i>P</i>_<i>GAL1</i><i>-GFP</i>) was used to monitor the <i>P</i>_<i>GAL1</i> induction.</p><p></p><p></p><p></p

Expression analysis of <i>sORF2</i>.

<p></p><p></p><p></p><p>RNAseq analysis of the <i>sORF2</i> region of the strain BY4741 with the control vector (pTOWug2–836) and pTOW-Rear2. Parts of the detected reads are shown. The locations of <i>DIE2</i>, <i>sORF2</i>, and <i>SMI1</i> are indicated.</p><p></p><p></p><p>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 (pTOWug2–836). 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 SC—Ura 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.</p><p></p><p></p><p></p

Multicopy suppressors of growth inhibition after increasing the copy number in the <i>DIE2</i> 3′ fragment.

<p></p><p></p><p></p><p>Maximum growth rate of BY4741 cells that harbored both pTOW-Rear2 and the suppressor plasmids (pRS423-<i>UBP7</i> and pRS423-<i>PRM1</i>, and the empty vector, pRS423) in SC—Ura—His medium. The averages and standard deviations from six independent experiments are shown.</p><p></p><p></p><p>Growth curves of the BY4741 cells that harbored both pTOW-Rear2 and the suppressor plasmids in SC—Ura—His medium. One representative data is shown from each experiment.</p><p></p><p></p><p></p

Genes with higher expression levels in cells that harbored pTOW-Rear2 compared with the control cells.

<p>*<i>Saccharomyces</i> genome database: <a href="http://www.yeastgenome.org" target="_blank">http://www.yeastgenome.org</a></p><p>Genes with higher expression levels in cells that harbored pTOW-Rear2 compared with the control cells.</p

Plasmids used in this study.

<p>Plasmids used in this study.</p

Post-Translational Dosage Compensation Buffers Genetic Perturbations to Stoichiometry of Protein Complexes

<div><p>Understanding buffering mechanisms for various perturbations is essential for understanding robustness in cellular systems. Protein-level dosage compensation, which arises when changes in gene copy number do not translate linearly into protein level, is one mechanism for buffering against genetic perturbations. Here, we present an approach to identify genes with dosage compensation by increasing the copy number of individual genes using the genetic tug-of-war technique. Our screen of chromosome I suggests that dosage-compensated genes constitute approximately 10% of the genome and consist predominantly of subunits of multi-protein complexes. Importantly, because subunit levels are regulated in a stoichiometry-dependent manner, dosage compensation plays a crucial role in maintaining subunit stoichiometries. Indeed, we observed changes in the levels of a complex when its subunit stoichiometries were perturbed. We further analyzed compensation mechanisms using a proteasome-defective mutant as well as ribosome profiling, which provided strong evidence for compensation by ubiquitin-dependent degradation but not reduced translational efficiency. Thus, our study provides a systematic understanding of dosage compensation and highlights that this post-translational regulation is a critical aspect of robustness in cellular systems.</p></div

Complex subunits tend to be subjected to dosage compensation.

<p>(<b>A</b>) Western blots of subunits composed of the five complexes. The experiments were performed using the same method with the screening. TAP-tagged target proteins expressed from the genomic regions were detected with PAP. The dosage-compensated proteins identified from the screening are shown in bold letters. (<b>B</b>) Quantification of protein expressions of the subunit genes. (<b>C</b>) Quantification of mRNA expressions of the subunit genes. The mRNA level of each TAP-tagged target gene was measured as described above. Dashed line denotes the same expression level between the Multi and Single conditions. The RNase MRP and nuclear RNase P subunit genes were analyzed in three biological replicates, and the average fold changes ± s.d. were calculated relative to the Single condition. ND: not detected.</p

Translational efficiency of <i>POP5</i> is not changed during dosage compensation.

<p>(<b>A, B</b>) Scatter plots showing the changes in mRNA levels (<b>A</b>) and the translational efficiency (<b>B</b>) of the genome in the Pop5-TAP strain carrying multicopy plasmid pTOWug2-<i>POP5</i> grown in SC–Ura medium. The X-axes indicate the mRNA level of each gene obtained by RNA-seq (mean counts in RNA-seq). The translational efficiency of each gene was calculated by dividing the ribosome density by the mRNA level. The mean fold changes relative to the Single condition were calculated from two biological replicates. (<b>C</b>) Bar graph indicates the mRNA level and translational efficiency of <i>POP5</i> shown in Fig 3A and 3B.</p