The determination of the exact location of a protein in the cell is essential to the understanding of biological processes. Here, we report for the first time the visualization of a protein of interest in Saccharomyces cerevisiae using focused ion beam scanning electron microscopy (FIB-SEM). As a proof of concept, the integral endoplasmic reticulum (ER) membrane protein Erg11 has been C-terminally tagged with APEX2, which is an engineered peroxidase that catalyzes an electron-dense deposition of 3,3'-diaminobenzidine (DAB), as such marking the location of the fused protein of interest in electron microscopic images. As DAB is unable to cross the yeast cell wall to react with APEX2, cell walls have been partly removed by the formation of spheroplasts. This has resulted in a clear electron-dense ER signal for the Erg11 protein using FIB-SEM. With this study, we have validated the use of the APEX2 tag for visualization of yeast proteins in electron microscopy. Furthermore, we have introduced a methodology that enables precise and three-dimensional (3D) localization studies in yeast, with nanometer resolution and without the need for antibody staining. Because of these properties, the described technique can offer valuable information on the molecular functions of studied proteins.IMPORTANCE With this study, we have validated the use of the APEX2 tag to define the localization of proteins in the model yeast S. cerevisiae As such, FIB-SEM can identify the exact 3D location of a protein of interest in the cell with nanometer-scale resolution. Such detailed imaging could provide essential information on the elucidation of various biological processes. APEX2, which adds electron density to a fused protein of interest upon addition of the substrate DAB, originally was used in mammalian studies. With this study, we expand its use to protein localization studies in one of the most important models in molecular biology.

中文翻译：

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使用聚焦离子束扫描电子显微镜在酿酒酵母中对APEX2标签的Erg11进行三维可视化。

确定蛋白质在细胞中的确切位置对于理解生物学过程至关重要。在这里，我们首次报道了使用聚焦离子束扫描电子显微镜（FIB-SEM）在酿酒酵母中感兴趣的蛋白质的可视化。作为概念的证明，完整的内质网（ER）膜蛋白Erg11已在C端标记有APEX2，APEX2是一种工程化的过氧化物酶，可催化3,3'-二氨基联苯胺（DAB）的电子致密沉积。在电子显微图像中标记感兴趣的融合蛋白的位置。由于DAB无法穿过酵母细胞壁与APEX2反应，因此通过原生质球的形成已部分去除了细胞壁。使用FIB-SEM得出Erg11蛋白的清晰电子密度ER信号。通过这项研究，我们已经验证了APEX2标签在电子显微镜中用于可视化酵母蛋白的有效性。此外，我们介绍了一种方法，该方法可在酵母中进行精确的三维（3D）定位研究，具有纳米分辨率，并且不需要抗体染色。由于这些特性，所描述的技术可以为所研究蛋白质的分子功能提供有价值的信息。重要事项通过这项研究，我们已经验证了APEX2标签在模型酵母中对蛋白质定位的有效性。 FIB-SEM可以纳米级的分辨率识别目标蛋白质在细胞中的确切3D位置。这样的详细成像可以提供有关阐明各种生物学过程的基本信息。APEX2，当加入底物DAB时，它在感兴趣的融合蛋白中增加了电子密度，最初用于哺乳动物研究。通过这项研究，我们将其用于分子生物学最重要模型之一的蛋白质定位研究中。