Toxicity of the model protein 3×GFP arises from degradation overload, not from aggregate formation.

Although protein aggregation can cause cytotoxicity, such aggregates can also form to mitigate cytotoxicity from misfolded proteins, although the nature of these contrasting aggregates remains unclear. We previously found that overproduction (op) of a three green fluorescent protein-linked protein (3×GFP) induces giant aggregates and is detrimental to growth. Here, we investigated the mechanism of growth inhibition by 3×GFP-op using non-aggregative 3×MOX-op as a control in Saccharomyces cerevisiae. The 3×GFP aggregates were induced by misfolding, and 3×GFP-op had higher cytotoxicity than 3×MOX-op because it perturbed the ubiquitin-proteasome system. Static aggregates formed by 3×GFP-op dynamically trapped Hsp70 family proteins (Ssa1 and Ssa2 in yeast), causing the heat-shock response. Systematic analysis of mutants deficient in the protein quality control suggested that 3×GFP-op did not cause a critical Hsp70 depletion and aggregation functioned in the direction of mitigating toxicity. Artificial trapping of essential cell cycle regulators into 3×GFP aggregates caused abnormalities in the cell cycle. In conclusion, the formation of the giant 3×GFP aggregates itself is not cytotoxic, as it does not entrap and deplete essential proteins. Rather, it is productive, inducing the heat-shock response while preventing an overload to the degradation system.

Protein sorting upon exit from the endoplasmic reticulum dominates Golgi biogenesis in budding yeast.

Cells sense and control the number and quality of their organelles, but the underlying mechanisms of this regulation are not understood. Our recent research in the yeast Saccharomyces cerevisiae has shown that long acyl chain ceramides in the endoplasmic reticulum (ER) membrane and the lipid moiety of glycosylphosphatidylinositol (GPI) anchor determine the sorting of GPI-anchored proteins in the ER. Here, we show that a mutant strain, which produces shorter ceramides than the wild-type strain, displays a different count of Golgi cisternae. Moreover, deletions of proteins that remodel the lipid portion of GPI anchors resulted in an abnormal number of Golgi cisternae. Thus, our study reveals that protein sorting in the ER plays a critical role in maintaining Golgi biogenesis.

Demonstration of iodide-dependent UVA-triggered growth inhibition in Saccharomyces cerevisiae cells and identification of its suppressive molecules.

Upon white light illumination, the growth of the budding yeast Saccharomyces cerevisiae was extremely impaired only in the presence of iodide ions, but not fluoride, chloride and bromide ions. Action spectroscopy revealed that the maximum wavelength of the light is around at 373 nm, corresponding to the UVA region. Using a genetic approach, several genes, including OPY1, HEM1, and PAU11, were identified as suppressors of this growth inhibition. This iodide-dependent UVA-triggered growth inhibition method, along with its suppressive molecules, would be beneficial for understanding cell growth processes in eukaryotes and can be utilized for medium sterilization using UVA light.

Overexpression profiling reveals cellular requirements in the context of genetic backgrounds and environments.

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.

Massive expression of cysteine-containing proteins causes abnormal elongation of yeast cells by perturbing the proteasome.

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.

Identification of uncharacterized proteins potentially localized to mitochondria (UPMs) in Saccharomyces cerevisiae using a fluorescent protein unstable in the cytoplasm.

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.

Genetic profiling of protein burden and nuclear export overload.

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.

Exploring the Complexity of Protein-Level Dosage Compensation that Fine-Tunes Stoichiometry of Multiprotein Complexes.

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.

Comparative Gene Analysis Focused on Silica Cell Wall Formation: Identification of Diatom-Specific SET Domain Protein Methyltransferases.

Silica cell walls of diatoms have attracted attention as a source of nanostructured functional materials and have immense potential for a variety of applications. Previous studies of silica cell wall formation have identified numerous involved proteins, but most of these proteins are species-specific and are not conserved among diatoms. However, because the basic process of diatom cell wall formation is common to all diatom species, ubiquitous proteins and molecules will reveal the mechanisms of cell wall formation. In this study, we assembled de novo transcriptomes of three diatom species, Nitzschia palea, Achnanthes kuwaitensis, and Pseudoleyanella lunata, and compared protein-coding genes of five genome-sequenced diatom species. These analyses revealed a number of diatom-specific genes that encode putative endoplasmic reticulum-targeting proteins. Significant numbers of these proteins showed homology to silicanin-1, which is a conserved diatom protein that reportedly contributes to cell wall formation. These proteins also included a previously unrecognized SET domain protein methyltransferase family that may regulate functions of cell wall formation-related proteins and long-chain polyamines. Proteomic analysis of cell wall-associated proteins in N. palea identified a protein that is also encoded by one of the diatom-specific genes. Expression analysis showed that candidate genes were upregulated in response to silicon, suggesting that these genes play roles in silica cell wall formation. These candidate genes can facilitate further investigations of silica cell wall formation in diatoms.

N-terminal deletion of Swi3 created by the deletion of a dubious ORF YJL175W mitigates protein burden effect in S. cerevisiae.

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.

Development of an experimental method of systematically estimating protein expression limits in HEK293 cells.

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.

Yeast screening system reveals the inhibitory mechanism of cancer cell proliferation by benzyl isothiocyanate through down-regulation of Mis12.

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.

Estimating the protein burden limit of yeast cells by measuring the expression limits of glycolytic proteins.

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.

Genetic Analysis of Signal Generation by the Rgt2 Glucose Sensor of Saccharomyces cerevisiae.

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.

Assessing phagotrophy in the mixotrophic ciliate Paramecium bursaria using GFP-expressing yeast cells.

We used cells of the yeast Saccharomyces cerevisiae expressing green fluorescent protein (GFP) as fluorescently labelled prey to assess the phagocytic activities of the mixotrophic ciliate Paramecium bursaria, which harbours symbiotic Chlorella-like algae. Because of different fluorescence spectra of GFP and algal chlorophyll, ingested GFP-expressing yeast cells can be distinguished from endosymbiotic algal cells and directly counted in individual P. bursaria cells using fluorescence microscopy. By using GFP-expressing yeast cells, we found that P. bursaria altered ingestion activities under different physiological conditions, such as different growth phases or the presence/absence of endosymbionts. Use of GFP-expressing yeast cells allowed us to estimate the digestion rates of live prey of the ciliate. In contrast to the ingestion activities, the digestion rate within food vacuoles was not affected by the presence of endosymbionts, consistent with previous findings that food and perialgal vacuoles are spatially and functionally separated in P. bursaria. Thus, GFP-expressing yeast may provide a valuable tool to assess both ingestion and digestion activities of ciliates that feed on eukaryotic organisms.

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

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.

Cellular growth defects triggered by an overload of protein localization processes.

High-level expression of a protein localized to an intracellular compartment is expected to cause cellular defects because it overloads localization processes. However, overloads of localization processes have never been studied systematically. Here, we show that the expression levels of green fluorescent proteins (GFPs) with localization signals were limited to the same degree as a toxic misfolded GFP in budding yeast cells, and that their high-level expression caused cellular defects associated with localization processes. We further show that limitation of the exportin Crm1 determined the expression limit of GFP with a nuclear export signal. Although misfolding of GFP with a vesicle-mediated transport signal triggered endoplasmic reticulum stress, it was not the primary determinant of its expression limit. The precursor of GFP with a mitochondrial targeting signal caused a cellular defect. Finally, we estimated the residual capacities of localization processes. High-level expression of a localized protein thus causes cellular defects by overloading the capacities of localization processes.

Quantitative nature of overexpression experiments.

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.

Aneuploid proliferation defects in yeast are not driven by copy number changes of a few dosage-sensitive genes.

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.