The expression level and cytotoxicity of green fluorescent protein are modulated by an additional N-terminal sequence

Nucleotide and amino acid sequences at the N-terminus affect the expression level and cytotoxicity of proteins; however, their effects are not fully understood yet. Here, N-terminal 30 nucleotide/10 amino acid (N10) sequences that affect the expression level and cytotoxicity of a green fluorescent protein were systematically isolated in the budding yeast Saccharomyces cerevisiae. The expression per gene (EPG) and gene copy number limit (CNL) relationships were examined to assess the effects of the N10 sequence. The isolated N10 nucleotide sequences suggested that codon optimality is the major determinant of the protein expression level. A higher number of hydrophobic or cysteine residues in the N10 sequence seemed to increase the cytotoxicity of the protein. Therefore, a high frequency of specific amino acid residues in the outside of the main tertiary structure of proteins might not be preferable

Mathematical analysis of copy number variation of 2 μ-based plasmids in yeast cells

　Plasmids with the 2 μ plasmid origin are commonly-used in the genetic engineering of the budding yeast Saccharomyces cerevisiae. Intracellular copy numbers of 2 μ plasmids are different depending on the genes inserted into the plasmids. This difference is thought to occur from the difference in the growth efficiency （fitness） produced by the positive- and negative-selection biases of genes inserted in the plasmid. In this study, we made a mathematical model based on this assumption. Computational simulations of the model validated that copy numbers of the plasmids are rapidly settled depending on the fitness created by the gene on the plasmid. The copy number of a plasmid only contains a bias to keep the plasmid in a single copy became average 20copies per cell when the plasmid is randomly distributed, suggesting that no positive distribution mechanism is required for a plasmid to become multicopy

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. Author summary Quality control of multiprotein complexes is important for maintaining homeostasis in cellular systems that are based on functional complexes. Proper stoichiometry of multiprotein complexes is achieved by the balance between protein synthesis and degradation. Recent studies showed that translation efficiency tends to scale with stoichiometry of their subunits. On the other hand, although protein N-terminal acetylation- and ubiquitin-mediated proteolysis pathway is involved in selective degradation of excess subunits, it is unclear how widespread this pathway contributes to stoichiometry control due to the lack of a systematic investigation using endogenous proteins. To better understand the landscape of the stoichiometry control system, we examined genetic interactions between 18 subunits and 12 quality control factors (E3 ubiquitin ligases and N-acetyltransferases), in total 114 combinations. Our data suggest that N-acetyltransferases are partially responsible for stoichiometry control and that N-acetylation-independent pathway is also involved in selective degradation of excess subunits. Therefore, this study reveals the complexity and robustness of the stoichiometry control system. Further dissection of this complexity will help to understand the mechanisms buffering gene expression perturbations and shaping proteome stoichiometry

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.National Institutes of Health (U.S.) (GM056800

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 similar to 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

Plasmid Construction Using Recombination Activity in the Fission Yeast Schizosaccharomyces pombe

BACKGROUND: Construction of plasmids is crucial in modern genetic manipulation. As of now, the common method for constructing plasmids is to digest specific DNA sequences with restriction enzymes and to ligate the resulting DNA fragments with DNA ligase. Another potent method to construct plasmids, known as gap-repair cloning (GRC), is commonly used in the budding yeast Saccharomyces cerevisiae. GRC makes use of the homologous recombination activity that occurs within the yeast cells. Due to its flexible design and efficiency, GRC has been frequently used for constructing plasmids with complex structures as well as genome-wide plasmid collections. Although there have been reports indicating GRC feasibility in the fission yeast Schizosaccharomyces pombe, this species is not commonly used for GRC as systematic studies of reporting GRC efficiency in S. pombe have not been performed till date. METHODOLOGY/PRINCIPAL FINDINGS: We investigated GRC efficiency in S. pombe in this study. We first showed that GRC was feasible in S. pombe by constructing a plasmid that contained the LEU2 auxotrophic marker gene in vivo and showed sufficient efficiency with short homology sequences (>25 bp). No preference was shown for the sequence length from the cut site in the vector plasmid. We next showed that plasmids could be constructed in a proper way using 3 DNA fragments with 70% efficiency without any specific selections being made. The GRC efficiency with 3 DNA fragments was dramatically increased >95% in lig4Delta mutant cell, where non-homologous end joining is deficient. Following this approach, we successfully constructed plasmid vectors with leu1+, ade6+, his5+, and lys1+ markers with the low-copy stable plasmid pDblet as a backbone by applying GRC in S. pombe. CONCLUSIONS/SIGNIFICANCE: We concluded that GRC was sufficiently feasible in S. pombe for genome-wide gene functional analysis as well as for regular plasmid construction. Plasmids with different markers constructed in this research are available from NBRP-yeast (http://yeast.lab.nig.ac.jp/)

Evaluator of adaptability of S. cerevisiae to grape juice using the oversxpression profiling ADOPT method

The authors have recently developed the overexpression profiling ADOPT method. In the ADOPT method, yeast strains overexpressing most of the genes in the budding yeast Saccharomyces cerevisiae genome are mixed and competitively cultured, and the genes overexpressed in the enriched strains are systematically identified. Furthermore, the identified genes can be used to identify bottleneck factors that are necessary but lacking for growth of S. cerevisiae under given conditions. In our previous studies, we have identified bottlenecks in artificially created stress environments in the laboratory, but in this study, we used grape juice for winemaking as an example to see if industrial bottlenecks can be identified. ADOPT experiments with sulfite-added grape juice used in conventional winemaking resulted in a strong enrichment of strains overexpressing the sulfite pump SSU1 and its transcription factor FZF1. Since enhancement of SSU1 function is known to occur in wine yeast acclimation, ADOPT was also shown to be useful in the search for industrial bottlenecks. On the other hand, no genes were strongly enriched by ADOPT in grape juice without sulfite addition, suggesting that grape juice is a balanced medium with few bottlenecks for S. cerevisiae growth.筆者らは最近，過剰発現プロファイリングADOPT 法を開発した．ADOPT 法では，出芽酵母Saccharomyces cerevisiae ゲノムのほとんどの遺伝子をそれぞれ過剰発現する酵母株を混合・競合培養し，その過程で濃縮されてきた株が過剰発現している遺伝子を体系的に同定する．さらに同定された遺伝子をたよりに，S. cerevisiae の与えられた条件での増殖に必要だが欠落しているボトルネック因子を明らかにできる．これまでの研究では，実験室で人為的に構築したストレス環境でのボトルネックの同定を行ってきたが，本研究では産業上のボトルネックを明らかにできるかをワイン醸造用のブドウ果汁を例として検証した．通常のワイン醸造に用いられる亜硫酸添加ブドウ果汁でのADOPT 実験は，亜硫酸ポンプSSU1とその転写因子FZF1の過剰発現株が強く濃縮された．SSU1機能の強化はワイン用酵母の馴養でも起きることが知られていることから，産業上のボトルネックを探索する際にもADOPT が有効であることが示された．一方，亜硫酸添加のないブドウ果汁ではADOPT で強く濃縮された遺伝子は見られず，ブドウ果汁はS. cerevisiae の増殖にとってボトルネックの少ないバランスのとれた培地であることが示唆された