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APEX2-mediated proximity labeling resolves protein networks in Saccharomyces cerevisiae cells.
The FEBS Journal ( IF 5.5 ) Pub Date : 2019-08-12 , DOI: 10.1111/febs.15007
Birgit Singer-Krüger 1 , Theresa Fröhlich 1 , Mirita Franz-Wachtel 2 , Nicolas Nalpas 2 , Boris Macek 2 , Ralf-Peter Jansen 1
Affiliation  

Enzyme-catalyzed proximity labeling (PL) with the engineered ascorbate peroxidase APEX2 is a novel approach to map organelle compartmentalization and protein networks in living cells. Current procedures developed for mammalian cells do not allow delivery of the cosubstrate, biotin-phenol, into living yeast cells. Here, we present a new method based on semipermeabilized yeast cells. Combined with stable isotope labeling by amino acids in cell culture (SILAC), we demonstrate proteomic mapping of a membrane-enclosed and a semiopen compartment, the mitochondrial matrix and the nucleus. APEX2 PL revealed nuclear proteins that were previously not identified by conventional techniques. One of these, the Yer156C protein, is highly conserved but of unknown function. Its human ortholog, melanocyte proliferating gene 1, is linked to developmental processes and dermatological diseases. A first characterization of the Yer156C neighborhood reveals an array of proteins linked to proteostasis and RNA binding. Thus, our approach establishes APEX2 PL as another powerful tool that complements the methods palette for the model system yeast.

中文翻译:

APEX2介导的邻近标记可解析酿酒酵母细胞中的蛋白质网络。

工程抗坏血酸过氧化物酶APEX2的酶催化邻近标记(PL)是一种映射活细胞中细胞器区室化和蛋白质网络的新方法。针对哺乳动物细胞开发的当前程序不允许将共底物生物素-酚传递到活酵母细胞中。在这里,我们提出了一种基于半透化酵母细胞的新方法。结合细胞培养物中氨基酸的稳定同位素标记(SILAC),我们证明了膜封闭和半开放隔室,线粒体基质和细胞核的蛋白质组学定位。APEX2 PL揭示了以前无法通过常规技术鉴定的核蛋白。其中之一,Yer156C蛋白,高度保守,但功能未知。它的人类直系同源物,黑色素细胞增殖基因1,与发育过程和皮肤病有关。Yer156C邻域的第一个特征显示了一系列与蛋白稳定和RNA结合相关的蛋白质。因此,我们的方法将APEX2 PL建立为另一个强大的工具,可以补充模型系统酵母的方法选项。
更新日期:2020-01-21
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