Authorship：Last author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Public Library of Science (PLoS)  

Overexpression can help life adapt to stressful environments, making an examination of overexpressed genes valuable for understanding stress tolerance mechanisms. However, a systematic study of genes whose overexpression is functionally adaptive (GOFAs) under stress has yet to be conducted. We developed a new overexpression profiling method and systematically identified GOFAs in Saccharomyces cerevisiae under stress (heat, salt, and oxidative). Our results show that adaptive overexpression compensates for deficiencies and increases fitness under stress, like calcium under salt stress. We also investigated the impact of different genetic backgrounds on GOFAs, which varied among three S. cerevisiae strains reflecting differing calcium and potassium requirements for salt stress tolerance. Our study of a knockout collection also suggested that calcium prevents mitochondrial outbursts under salt stress. Mitochondria-enhancing GOFAs were only adaptive when adequate calcium was available and non-adaptive when calcium was deficient, supporting this idea. Our findings indicate that adaptive overexpression meets the cell’s needs for maximizing the organism’s adaptive capacity in the given environment and genetic context.

DOI： 10.1371/journal.pgen.1010732

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Authorship：Last author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press (OUP)  

Abstract

The enhanced green fluorescent protein (EGFP) is considered to be a harmless protein because the critical expression level that causes growth defects is higher than that of other proteins. Here, we found that overexpression of EGFP, but not a glycolytic protein Gpm1, triggered the cell elongation phenotype in the budding yeast Saccharomyces cerevisiae. By the morphological analysis of the cell overexpressing fluorescent protein and glycolytic enzyme variants, we revealed that cysteine content was associated with the cell elongation phenotype. The abnormal cell morphology triggered by overexpression of EGFP was also observed in the fission yeast Schizosaccharomyces pombe. Overexpression of cysteine-containing protein was toxic, especially at high-temperature, while the toxicity could be modulated by additional protein characteristics. Investigation of protein aggregate formation, morphological abnormalities in mutants, and transcriptomic changes that occur upon overexpression of EGFP variants suggested that perturbation of the proteasome by the exposed cysteine of the overexpressed protein causes cell elongation. Overexpression of proteins with relatively low folding properties, such as EGFP, was also found to promote the formation of SHOTA (Seventy kDa Heat shock protein-containing, Overexpression-Triggered Aggregates), an intracellular aggregate that incorporates Hsp70/Ssa1, which induces a heat shock response, while it was unrelated to cell elongation. Evolutionary analysis of duplicated genes showed that cysteine toxicity may be an evolutionary bias to exclude cysteine from highly expressed proteins. The overexpression of cysteine-less moxGFP, the least toxic protein revealed in this study, would be a good model system to understand the physiological state of protein burden triggered by ultimate overexpression of harmless proteins.

DOI： 10.1093/g3journal/jkac106

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

Eukaryotic cells are composed of organelles, and each organelle contains proteins that play a role in its function. Therefore, the localization of a protein, especially to organelles, is a clue to infer the function of that protein. In this study, we attempted to identify novel mitochondrially localized proteins in the budding yeast Saccharomyces cerevisiae using a fluorescent protein (GFPdeg) that is rapidly degraded in the cytoplasm. Of the budding yeast proteins predicted to localize to mitochondria by the prediction tool Deeploc-1.0, those with known mitochondrial localization or functional relevance were eliminated, and 95 proteins of unknown function were selected as candidates for analysis. By forced expression of GFPdeg fusion proteins with these proteins and observation of their localization, we identified 35 uncharacterized proteins potentially localized to mitochondria (UPMs) including 8 previously identified proteins that localize to mitochondria. Most of these had no N-terminal mitochondrial localization signal and were evolutionarily young "emerging genes" that exist only in S. cerevisiae. Some of these genes were found to be upregulated during the postdiauxic shift phase when mitochondria are being developed, suggesting that they are actually involved in some mitochondrial function.

DOI： 10.1002/yea.3685

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：eLife Sciences Publications, Ltd  

Overproduction (op) of proteins triggers cellular defects. One of the consequences of overproduction is the protein burden/cost, which is produced by an overloading of the protein synthesis process. However, the physiology of cells under a protein burden is not well characterized. We performed genetic profiling of protein burden by systematic analysis of genetic interactions between GFP-op, surveying both deletion and temperature-sensitive mutants in budding yeast. We also performed genetic profiling in cells with overproduction of triple-GFP (tGFP), and the nuclear export signal-containing tGFP (NES-tGFP). The mutants specifically interacted with GFP-op were suggestive of unexpected connections between actin-related processes like polarization and the protein burden, which was supported by morphological analysis. The tGFP-op interactions suggested that this protein probe overloads the proteasome, whereas those that interacted with NES-tGFP involved genes encoding components of the nuclear export process, providing a resource for further analysis of the protein burden and nuclear export overload.

DOI： 10.7554/elife.54080

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Other Link： https://cdn.elifesciences.org/articles/54080/elife-54080-v1.xml

Authorship：Last author   Publishing type：Research paper (scientific journal)   Publisher：Public Library of Science (PLoS)  

Proper control of gene expression levels upon various perturbations is a fundamental aspect of cellular robustness. Protein-level dosage compensation is one mechanism buffering perturbations to stoichiometry of multiprotein complexes through accelerated proteolysis of unassembled subunits. Although N-terminal acetylation- and ubiquitin-mediated proteasomal degradation by the Ac/N-end rule pathway enables selective compensation of excess subunits, it is unclear how widespread this pathway contributes to stoichiometry control. Here we report that dosage compensation depends only partially on the Ac/N-end rule pathway. Our analysis of genetic interactions between 18 subunits and 12 quality control factors in budding yeast demonstrated that multiple E3 ubiquitin ligases and N-acetyltransferases are involved in dosage compensation. We find that N-acetyltransferases-mediated compensation is not simply predictable from N-terminal sequence despite their sequence specificity for N-acetylation. We also find that the compensation of Pop3 and Bet4 is due in large part to a minor N-acetyltransferase NatD. Furthermore, canonical NatD substrates histone H2A/H4 were compensated even in its absence, suggesting N-acetylation-independent stoichiometry control. Our study reveals the complexity and robustness of the stoichiometry control system.

DOI： 10.1371/journal.pgen.1009091

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Extreme overproduction of gratuitous proteins can overload cellular protein production resources, leading to growth defects, a phenomenon known as the protein burden/cost effect. Genetic screening in the budding yeast Saccharomyces cerevisiae has isolated several dubious ORFs whose deletions mitigated the protein burden effect, but individual characterization thereof has yet to be delineated. We found that deletion of the YJL175W ORF yielded an N-terminal deletion of Swi3, a subunit of the SWI/SNF chromatin remodeling complex, and partial loss of function of Swi3. The deletion mutant showed a reduction in transcription of genes encoding highly expressed, secreted proteins and an overall reduction in translation. Mutations in the chromatin remodeling complex could thus mitigate the protein burden effect, likely by reallocating residual cellular resources used to overproduce proteins. This cellular state might also be related to cancer cells, as they frequently harbor mutations in the SWI/SNF complex.

DOI： 10.1038/s41598-020-66307-z

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Protein overexpression sometimes causes cellular defects, although the underlying mechanism is still unknown. A protein's expression limit, which triggers cellular defects, is a useful indication of the underlying mechanism. In this study, we developed an experimental method of estimating the expression limits of target proteins in the human embryonic kidney cell line HEK293 by measuring the proteins' expression levels in cells that survived after the high-copy introduction of plasmid DNA by which the proteins were expressed under a strong cytomegalovirus promoter. The expression limits of nonfluorescent target proteins were indirectly estimated by measuring the levels of green fluorescent protein (GFP) connected to the target proteins with the self-cleaving sequence P2A. The expression limit of a model GFP was ~5.0% of the total protein, and sustained GFP overexpression caused cell death. The expression limits of GFPs with mitochondria-targeting signals and endoplasmic reticulum localization signals were 1.6% and 0.38%, respectively. The expression limits of four proteins involved in vesicular trafficking were far lower compared to a red fluorescent protein. The protein expression limit estimation method developed will be valuable for defining toxic proteins and consequences of protein overexpression.

DOI： 10.1038/s41598-020-61646-3

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Authorship：Lead author,　Last author,　Corresponding author   Publishing type：Research paper (scientific journal)  

DOI： 10.3934/BIOPHY.2020010

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Language：English   Publishing type：Research paper (scientific journal)  

Benzyl isothiocyanate (BITC) is a naturally-occurring isothiocyanate derived from cruciferous vegetables. BITC has been reported to inhibit the proliferation of various cancer cells, which is believed to be important for the inhibition of tumorigenesis. However, the detailed mechanisms of action remain unclear. In this study, we employed a budding yeast Saccharomyces cerevisiae as a model organism for screening. Twelve genes including MTW1 were identified as the overexpression suppressors for the antiproliferative effect of BITC using the genome-wide multi-copy plasmid collection for S. cerevisiae. Overexpression of the kinetochore protein Mtw1 counteracts the antiproliferative effect of BITC in yeast. The inhibitory effect of BITC on the proliferation of human colon cancer HCT-116 cells was consistently suppressed by the overexpression of Mis12, a human orthologue of Mtw1, and enhanced by the knockdown of Mis12. We also found that BITC increased the phosphorylated and ubiquitinated Mis12 level with consequent reduction of Mis12, suggesting that BITC degrades Mis12 through an ubiquitin-proteasome system. Furthermore, cell cycle analysis showed that the change in the Mis12 level affected the cell cycle distribution and the sensitivity to the BITC-induced apoptosis. These results provide evidence that BITC suppresses cell proliferation through the post-transcriptional regulation of the kinetochore protein Mis12.

DOI： 10.1038/s41598-019-45248-2

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

The ultimate overexpression of a protein could cause growth defects, which are known as the protein burden. However, the expression limit at which the protein-burden effect is triggered is still unclear. To estimate this limit, we systematically measured the overexpression limits of glycolytic proteins in Saccharomyces cerevisiae. The limits of some glycolytic proteins were up to 15% of the total cellular protein. These limits were independent of the proteins' catalytic activities, a finding that was supported by an in silico analysis. Some proteins had low expression limits that were explained by their localization and metabolic perturbations. The codon usage should be highly optimized to trigger the protein-burden effect, even under strong transcriptional induction. The S-S-bond-connected aggregation mediated by the cysteine residues of a protein might affect its expression limit. Theoretically, only non-harmful proteins could be expressed up to the protein-burden limit. Therefore, we established a framework to distinguish proteins that are harmful and non-harmful upon overexpression.

DOI： 10.7554/eLife.34595

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Other Link： http://orcid.org/0000-0001-7638-3640

Language：English   Publishing type：Research paper (scientific journal)  

The yeast S. cerevisiae senses glucose through Snf3 and Rgt2, transmembrane proteins that generate an intracellular signal in response to glucose that leads to inhibition of the Rgt1 transcriptional repressor and consequently to derepression of HXT genes encoding glucose transporters. Snf3 and Rgt2 are thought to be glucose receptors because they are similar to glucose transporters. In contrast to glucose transporters, they have unusually long C-terminal tails that bind to Mth1 and Std1, paralogous proteins that regulate function of the Rgt1 transcription factor. We show that the C-terminal tail of Rgt2 is not responsible for its inability to transport glucose. To gain insight into how the glucose sensors generate an intracellular signal, we identified RGT2 mutations that cause constitutive signal generation. Most of the mutations alter evolutionarily-conserved amino acids in the transmembrane spanning regions of Rgt2 that are predicted to be involved in maintaining an outward-facing conformation or to be in the substrate binding site. Our analysis of these mutations suggests they cause Rgt2 to adopt inward-facing or occluded conformations that generate the glucose signal. These results support the idea that Rgt2 and Snf3 are glucose receptors that signal in response to binding of extracellular glucose and inform the basis of their signaling.

DOI： 10.1534/g3.118.200338

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1093/femsle/fnx117

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1371/journal.pgen.1006554

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Other Link： http://orcid.org/0000-0001-7638-3640

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1038/srep31774

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Overexpression experiments are sometimes considered as qualitative experiments designed to identify novel proteins and study their function. However, in order to draw conclusions regarding protein overexpression through association analyses using large-scale biological data sets, we need to recognize the quantitative nature of overexpression experiments. Here I discuss the quantitative features of two different types of overexpression experiment: absolute and relative. I also introduce the four primary mechanisms involved in growth defects caused by protein overexpression: resource overload, stoichiometric imbalance, promiscuous interactions, and pathway modulation associated with the degree of overexpression.

DOI： 10.1091/mbc.E15-07-0512

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Language：English   Publishing type：Research paper (scientific journal)  

Aneuploidy-the gain or loss of one or more whole chromosome-typically has an adverse impact on organismal fitness, manifest in conditions such as Down syndrome. A central question is whether aneuploid phenotypes are the consequence of copy number changes of a few especially harmful genes that may be present on the extra chromosome or are caused by copy number alterations of many genes that confer no observable phenotype when varied individually. We used the proliferation defect exhibited by budding yeast strains carrying single additional chromosomes (disomes) to distinguish between the "few critical genes" hypothesis and the "mass action of genes" hypothesis. Our results indicate that subtle changes in gene dosage across a chromosome can have significant phenotypic consequences. We conclude that phenotypic thresholds can be crossed by mass action of copy number changes that, on their own, are benign.

DOI： 10.1101/gad.261743.115

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Other Link： http://orcid.org/0000-0001-7638-3640

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

In a previous study, we found an unknown element that caused growth inhibition after its copy number increased in the 3' 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.

DOI： 10.1371/journal.pone.0120678

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DOI： 10.1186/1752-0509-8-2

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：Springer Verlag  

DOI： 10.1007/978-3-319-10398-3_14

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DOI： 10.1038/NCHEMBIO.1366

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DOI： 10.1021/sb300116y

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DOI： 10.1101/gr.146662.112

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DOI： 10.2116/analsci.29.367

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DOI： 10.1371/journal.pone.0073319

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DOI： 10.1039/c2mb25100k

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DOI： 10.1002/cbic.201100798

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DOI： 10.1038/msb.2011.91

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DOI： 10.1038/msb.2010.73

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DOI： 10.1371/journal.pgen.1000919

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DOI： 10.1371/journal.pone.0009652

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DOI： 10.1371/journal.pgen.0020111

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DOI： 10.1128/EC.5.1.167-173.2006

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Language：English   Publishing type：Research paper (international conference proceedings)  

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DOI： 10.1016/j.jtcvs.2005.02.040

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DOI： 10.1073/pnas.0305901101

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DOI： 10.1101/gad.884001

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DOI： 10.1002/(SICI)1097-0061(199904)15:6<481::AID-YEA391>3.0.CO;2-M

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1093/nar/23.4.595

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Authorship：Lead author,　Last author,　Corresponding author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：Biophysical Society of Japan  

DOI： 10.2142/biophys.62.134

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

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Language：Japanese   Publisher：金原一郎記念医学医療振興財団 ; 1949-  

DOI： 10.11477/mf.2425200762

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Language：Japanese   Publisher：日本生物科学者協会 ; 1949-  

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Language：Japanese   Publisher：Japan Society for Bioscience, Biotechnology, and Agrochemistry  

細胞の機能は数千種類のタンパク質が協調的に働くことで達成される．それぞれのタンパク質の発現量はどのように決まっているのだろうか，またその量が変化したときに何が起きるのだろうか？ 本稿では，主に酵母を対象として，近年のオーミックスデータや筆者らのタンパク質発現限界のシステマティックな測定結果などを通じて，細胞がタンパク質発現リソースをどのように配分しているのか，細胞がどれくらいのタンパク質発現のキャパシティをもっているのかという視点で細胞を眺めてみたい．なお，本稿のデータや図の一部は，最近筆者が執筆した文献1から引用している．詳細な内容についてはそれも合わせて参照していただきたい．

DOI： 10.1271/kagakutoseibutsu.54.555

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Language：Japanese  

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Language：Japanese   Publisher：Japanese Proteomics Society (Japan Human Proteome Organisation)  

DOI： 10.14889/jhupo.2014.0.32.0

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Language：Japanese   Publisher：学研メディカル秀潤社 ; 1982-  

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Language：Japanese   Publisher：学研メディカル秀潤社 ; 1982-  

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.53.323

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Language：Japanese   Publisher：日本農芸化学会  

近年，天然物の生合成遺伝子の情報をもとに，有用天然物を獲得する取り組みが行なわれるようになってきた．中でも植物および真菌といった真核生物から単離された生物活性物質の生物合成に関する報告がなされてきた．ここでは，特に真核生物由来の天然物生合成遺伝子を出芽酵母の異種発現系を用いて発現させ，有用物質の生産に挑戦する取り組みについて解説する．

DOI： 10.1271/kagakutoseibutsu.50.163

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Language：Japanese   Publisher：社団法人 日本農芸化学会  

DOI： 10.1271/kagakutoseibutsu.47.269

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Language：Japanese   Publisher：東京化学同人  

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DOI： 10.1371/journal.pgen.0020218

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Language：Japanese   Publisher：システム制御情報学会  

DOI： 10.11509/isciesci.50.8_309

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Language：Japanese   Publisher：バイオインダストリー協会  

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