A Validated Set of Fluorescent-Protein-Based Markers for Major Organelles in Yeast (Saccharomyces cerevisiae)
A Validated Set of Fluorescent-Protein-Based Markers for Major Organelles in Yeast (Saccharomyces cerevisiae)
复制标题
一组经过验证的酵母（酿酒酵母）主要细胞器的基于荧光蛋白的标记
DOI：
10.1128/mbio.01691-19
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发表时间：
2019-09-01
期刊：
影响因子：
6.4
通讯作者：
Xie, Zhiping
中科院分区：
文献类型：
--
作者：
Zhu, Jing;Zhang, Zheng-Tan;Xie, Zhiping
摘要真核细胞具有以膜为基础的细胞器为特征的内部组织的基本模式。荧光蛋白的使用极大地促进了细胞器动力学和蛋白质运输的活细胞成像。这种方法的一个主要限制是FP与靶蛋白的融合可以并且经常损害靶蛋白的功能并改变其亚细胞定位。获得所需融合构建体的优化过程可能是耗时的或甚至是不成功的。在这项工作中，我们着手提供一套有效的基于FP的标记，主要细胞器的芽殖酵母（酿酒酵母）。在构建的160多个质粒中，我们提出了最后一组42个质粒，这些质粒的建议得到了细致评估的支持。该工具集包括三种颜色（绿色、红色和蓝色），涵盖内质网（ER）、细胞核、高尔基体、核内体、空泡、线粒体、过氧化物酶体和脂滴。通过系统的交叉比较和定量建立标记物的保真度。进行功能测定以检查标志物表达对线粒体和过氧化物酶体的分泌途径、内吞途径和代谢活性的影响。与此同时，我们的工作构成了在这个模式生物的细胞器身份的重新评估。我们的数据支持的认识，“晚期高尔基体”和“早期内体”，两个看似不同的术语，表示在酵母中的相同的隔间。相反，所有其他细胞器可以在传统光学显微镜的分辨率下彼此视觉分离，并且定量结果证明它们作为不同实体的分类是合理的。细胞含有复杂的内部结构。对于真核细胞，就像我们体内的细胞一样，内部空间被划分为膜结合的细胞器，每个细胞器都有专门的功能。通常，人们需要可视化细胞器来理解复杂的细胞过程。在这里，我们提供了一套有效的荧光蛋白为基础的标志物的主要细胞器在芽殖酵母。酵母是研究真核生物共有的基本机制时常用的模型。荧光蛋白由细胞自身产生，避免了昂贵的化学染料的需要。通过广泛的交叉比较，我们确保我们的每一个标记物都标记并且只标记预期的细胞器。我们还仔细检查了我们的标记物的存在是否对细胞的功能有任何负面影响，并没有发现。我们的工作还有助于回答一个相关的问题：我们看到的结构真的是我们认为的那样吗？
ABSTRACT Eukaryotic cells share a basic scheme of internal organization featuring membrane-based organelles. The use of fluorescent proteins (FPs) greatly facilitated live-cell imaging of organelle dynamics and protein trafficking. One major limitation of this approach is that the fusion of an FP to a target protein can and often does compromise the function of the target protein and alter its subcellular localization. The optimization process to obtain a desirable fusion construct can be time-consuming or even unsuccessful. In this work, we set out to provide a validated set of FP-based markers for major organelles in the budding yeast (Saccharomyces cerevisiae). Out of over 160 plasmids constructed, we present a final set of 42 plasmids, the recommendations for which are backed up by meticulous evaluations. The tool set includes three colors (green, red, and blue) and covers the endoplasmic reticulum (ER), nucleus, Golgi apparatus, endosomes, vacuoles, mitochondria, peroxisomes, and lipid droplets. The fidelity of the markers was established by systematic cross-comparison and quantification. Functional assays were performed to examine the impact of marker expression on the secretory pathway, endocytic pathway, and metabolic activities of mitochondria and peroxisomes. Concomitantly, our work constitutes a reassessment of organelle identities in this model organism. Our data support the recognition that “late Golgi” and “early endosomes,” two seemingly distinct terms, denote the same compartment in yeast. Conversely, all other organelles can be visually separated from each other at the resolution of conventional light microscopy, and quantification results justify their classification as distinct entities. IMPORTANCE Cells contain elaborate internal structures. For eukaryotic cells, like those in our bodies, the internal space is compartmentalized into membrane-bound organelles, each tasked with specialized functions. Oftentimes, one needs to visualize organelles to understand a complex cellular process. Here, we provide a validated set of fluorescent protein-based markers for major organelles in budding yeast. Yeast is a commonly used model when investigating basic mechanisms shared among eukaryotes. Fluorescent proteins are produced by cells themselves, avoiding the need for expensive chemical dyes. Through extensive cross-comparison, we make sure that each of our markers labels and only labels the intended organelle. We also carefully examined if the presence of our markers has any negative impact on the functionality of the cells and found none. Our work also helps answer a related question: are the structures we see really what we think they are?