We present a novel method to fluorescently label proteins, post-translationally, within live Saccharomycescerevisiae. The premise underlying this work is that fluorescent protein (FP) tags are less disruptive to normal processing and function when they are attached post-translationally, because target proteins are allowed to fold properly and reach their final subcellular location before being labeled. We accomplish this post-translational labeling by expressing the target protein fused to a short peptide tag (SpyTag), which is then covalently labeled in situ by controlled expression of an open isopeptide domain (SpyoIPD, a more stable derivative of the SpyCatcher protein) fused to an FP. The formation of a covalent bond between SpyTag and SpyoIPD attaches the FP to the target protein. We demonstrate the general applicability of this strategy by labeling several yeast proteins. Importantly, we show that labeling the membrane protein Pma1 in this manner avoids the mislocalization and growth impairment that occur when Pma1 is genetically fused to an FP. We also demonstrate that this strategy enables a novel approach to spatiotemporal tracking in single cells and we develop a Bayesian analysis to determine the protein's turnover time from such data.

中文翻译：

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一种在活细胞中翻译后标记蛋白质以进行荧光成像和跟踪的新方法。

我们提出了一种在活酿酒酵母中翻译后荧光标记蛋白质的新方法。这项工作的前提是荧光蛋白 (FP) 标签在翻译后附着时对正常加工和功能的干扰较小，因为目标蛋白在被标记之前可以正确折叠并到达其最终的亚细胞位置。我们通过表达与短肽标签 (SpyTag) 融合的目标蛋白来完成翻译后标记，然后通过融合的开放异肽结构域（SpyoIPD，SpyCatcher 蛋白的更稳定衍生物）的受控表达对其进行原位共价标记到 FP。SpyTag 和 SpyoIPD 之间形成共价键，将 FP 连接到目标蛋白上。我们通过标记几种酵母蛋白来证明该策略的普遍适用性。重要的是，我们表明以这种方式标记膜蛋白 Pma1 可以避免 Pma1 与 FP 基因融合时发生的错误定位和生长障碍。我们还证明，这种策略能够提供一种在单细胞中进行时空跟踪的新方法，并且我们开发了贝叶斯分析来根据这些数据确定蛋白质的周转时间。