Folding Latency of Fluorescent Proteins Affects the Mitochondrial Localization of Fusion Proteins
Folding Latency of Fluorescent Proteins Affects the Mitochondrial Localization of Fusion Proteins
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DOI：
10.1247/csf.19028
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发表时间：
2019-01-01
影响因子：
1.5
通讯作者：
Ohba, Yusuke
中科院分区：
文献类型：
--
作者：
Kashiwagi, Sayaka;Fujioka, Yoichiro;Ohba, Yusuke
荧光蛋白的发现彻底改变了细胞生物学。靶向序列与FPs的融合使得能够研究细胞器官及其动力学;然而，偶尔，这种荧光融合蛋白（FPs）表现出与天然蛋白不同的行为。在这里，我们构建了一个包含不同细胞器标记物的颜色托盘，并发现当与某些类型的FP融合时，靶向线粒体的FFP被错误定位。这种FP包括维多利亚水母绿色FP（avGFP）的几种变体和红色FP的单体变体。由于错误定位的FFP包括具有更快成熟或折叠突变的FP，因此成熟速率的增加可能会阻止其预期的定位。事实上，当我们重新引入氨基酸取代，使FP序列等同于温和型avGFP，FFP定位到线粒体显着增强。此外，类似的氨基酸取代改善了GFP的pH敏感性变体pHluorin的定位及其监测线粒体基质中pH变化的能力。我们的研究结果表明，选择最大化FFP功能的FP的重要性。
The discovery of fluorescent proteins (FPs) has revolutionized cell biology. The fusion of targeting sequences to FPs enables the investigation of cellular organdies and their dynamics; however, occasionally, such fluorescent fusion proteins (FFPs) exhibit behavior different from that of the native proteins. Here, we constructed a color pallet comprising different organelle markers and found that FFPs targeted to the mitochondria were mislocalized when fused to certain types of FPs. Such FPs included several variants of Aequorea victoria green FP (avGFP) and a monomeric variant of the red FP. Because the FFPs that are mislocalized include FPs with faster maturing or folding mutations, the increase in the maturation rate is likely to prevent their expected localization. Indeed, when we reintroduced amino acid substitutions so that the FP sequences were equivalent to that of mild-type avGFP, FFP localization to the mitochondria was significantly enhanced. Moreover, similar amino acid substitutions improved the localization of mitochondria-targeted pHluorin, which is a pH-sensitive variant of GFP, and its capability to monitor pH changes in the mitochondrial matrix. Our fmdings demonstrate the importance of selecting FPs that maximize FFP function.