Evaluation of the lower protein limit in the budding yeast Saccharomyces cerevisiae using TIPI-gTOW.
Evaluation of the lower protein limit in the budding yeast Saccharomyces cerevisiae using TIPI-gTOW.
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DOI：
10.1186/1752-0509-8-2
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发表时间：
2014-01-07
影响因子：
--
通讯作者：
Moriya H
中科院分区：
文献类型：
--
作者：
Sasabe M;Shintani S;Kintaka R;Kaizu K;Makanae K;Moriya H
确定细胞内参数（例如蛋白质表达）的允许限制为检查稳健性提供了重要信息。在本研究中，我们利用TEV蛋白酶介导的蛋白质不稳定性诱导（TIPI）与遗传拔河（gTOW）相结合，开发了一种测量蛋白质水平下限的方法。我们首先使用 ADE2 作为标记测试了该方法的可行性，然后分析了一些细胞周期调节因子以揭示遗传相互作用。使用TIPI-gTOW，我们成功构建了一种菌株，其中GFP-TDegFAde2的表达处于下限，通过加速TEV蛋白酶的降解足以支持-Ade条件下的细胞生长。我们还成功构建了 TIPI-gTOW 表达最低水平 GFP-TDegFCdc20 的菌株。使用该菌株，我们研究了细胞周期调节因子和CDC20之间的遗传相互作用，结果与之前确定的相互作用高度一致。将实验数据与数学模型的预测进行比较，揭示了一些当前模型中未实现的相互作用。 TIPI-gTOW 可用于估计不同条件（例如不同遗传背景和环境）下蛋白质下限的变化。 TIPI-gTOW 还可用于分析无法获得缺失突变体的必需基因的遗传相互作用。
Identifying permissible limits of intracellular parameters such as protein expression provides important information for examining robustness. In this study, we used the TEV protease-mediated induction of protein instability (TIPI) in combination with the genetic Tug-of-War (gTOW) to develop a method to measure the lower limit of protein level. We first tested the feasibility of this method using ADE2 as a marker and then analyzed some cell cycle regulators to reveal genetic interactions. Using TIPI-gTOW, we successfully constructed a strain in which GFP-TDegFAde2 was expressed at the lower limit, just sufficient to support cellular growth under the -Ade condition by accelerating degradation by TEV protease. We also succeeded in constructing a strain in which the minimal level of GFP-TDegFCdc20 was expressed by TIPI-gTOW. Using this strain, we studied genetic interactions between cell cycle regulators and CDC20, and the result was highly consistent with the previously identified interactions. Comparison of the experimental data with predictions of a mathematical model revealed some interactions that were not implemented into the current model. TIPI-gTOW is useful for estimating changes in the lower limit of a protein under different conditions, such as different genetic backgrounds and environments. TIPI-gTOW is also useful for analyzing genetic interactions of essential genes whose deletion mutants cannot be obtained.
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