The discovery of fluorescent proteins (FPs) has revolutionized cell biology. The fusion of targeting sequences to FPs enables the investigation of cellular organelles 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 wild-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 findings demonstrate the importance of selecting FPs that maximize FFP function.Key words: fluorescent protein, organelle, fusion protein, mitochondria.

中文翻译：

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荧光蛋白的折叠潜伏期影响融合蛋白的线粒体定位。

荧光蛋白（FPs）的发现彻底改变了细胞生物学。靶向序列与FP的融合使得能够研究细胞器及其动力学。但是，有时这种荧光融合蛋白（FFP）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色板，发现当与某些类型的FP融合时，针对线粒体的FFP定位不正确。这样的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。因为定位不正确的FFP包括成熟或折叠突变更快的FP，所以成熟速率的增加很可能会阻止其预期的定位。确实，当我们重新引入氨基酸取代以使FP序列与野生型avGFP相同时，FFP定位于线粒体的位置显着增强。此外，相似的氨基酸取代改善了针对线粒体的pHluorin（GFP的pH敏感变体）的定位，并改善了其监测线粒体基质中pH变化的能力。我们的发现证明了选择使FFP功能最大化的FP的重要性。关键词：荧光蛋白，细胞器，融合蛋白，线粒体。以及监测线粒体基质中pH值变化的能力。我们的发现表明选择使FFP功能最大化的FP的重要性。关键词：荧光蛋白，细胞器，融合蛋白，线粒体。以及监测线粒体基质中pH值变化的能力。我们的发现证明了选择使FFP功能最大化的FP的重要性。关键词：荧光蛋白，细胞器，融合蛋白，线粒体。